A novel CXCR4 antagonist IgG1 antibody (PF-06747143) for the treatment of hematologic malignancies

Abstract

The chemokine receptor CXCR4 is highly expressed and associated with poor prognosis in multiple malignancies. Upon engagement by its ligand, CXCL12, CXCR4 triggers intracellular signaling pathways that control trafficking of cells to tissues where the ligand is expressed, such as the bone marrow (BM). In hematologic cancers, CXCR4-driven homing of malignant cells to the BM protective niche is a key mechanism driving disease and therapy resistance. We developed a humanized CXCR4 immunoglobulin G1 (IgG1) antibody (Ab), PF-06747143, which binds to CXCR4 and inhibits CXCL12-mediated signaling pathways, as well as cell migration. In in vivo preclinical studies, PF-06747143 monotherapy rapidly and transiently mobilized cells from the BM into the peripheral blood. In addition, PF-06747143 effectively induced tumor cell death via its Fc constant region-mediated effector function. This Fc-mediated cell killing mechanism not only enhanced antitumor efficacy, but also played a role in reducing the duration of cell mobilization, when compared with an IgG4 version of the Ab, which does not have Fc-effector function. PF-06747143 treatment showed strong antitumor effect in multiple hematologic tumor models including non-Hodgkin lymphoma (NHL), acute myeloid leukemia (AML), and multiple myeloma (MM). Importantly, PF-06747143 synergized with standard-of-care agents in a chemoresistant AML patient-derived xenograft model and in an MM model. These findings suggest that PF-06747143 is a potential best-in-class anti-CXCR4 antagonist for the treatment of hematologic malignancies, including in the resistant setting. PF-06747143 is currently in phase 1 clinical trial evaluation (registered at www.clinicaltrials.gov as #NCT02954653).

Conflict of interest statement

Conflict-of-interest disclosure: All authors were employees of Pfizer Inc. at the time the work was performed. None of the authors received grants for this work.

Figures

Graphical abstract

Figure 1.

PF-06747143 blocks CXCL12-induced pathways. (A) cAMP assay was performed in CHO-K1 cells transfected with hCXCR4 (CHO-K1-CXCR4), incubated with CXCL12 at its EC80 (1.25 nM) and PF-06747143 or IgG1 control Ab. Experiments were performed in triplicates. Bars represent standard error of the mean (SEM). (B) Migration assay was performed in Ramos human NHL cells incubated in transwell chambers for 24 hours with PF-06747143 or IgG1 control Ab. CXCL12 (100 ng/mL) was used a chemoattractant in the bottom chamber. Data are shown as mean % migration inhibition. Experiment was performed in quadruplicates. Bars represent SEM.

Figure 2.

PF-06747143 induces malignant cell death by Fc-effector function activity. ADCC activity was evaluated by incubating 100 nM PF-06747143, m15-IgG1, m15-IgG4, rituximab, or respective negative control Ab’s for 4 hours in the presence of NK92 158V effector killer cells (effector:target cell ratio 10:1) with tumor target cells: Ramos (A) and MV4-11 and OPM-2 (B). Cell lysis was measured by ToxiLight bioluminescent cytotoxicity assay. Experiments were performed in quadruplicates. Bars represent SEM. CDC was assessed by incubating Daudi NHL target cells in the presence of 2.5% human complement for 4 hours. Experiments were performed in duplicates. Bars represent standard deviations. (C) Cells were treated with 33 nM of IgG1 control Ab, m15-IgG1, m15-IgG4, or rituximab. (D) Cells were treated with 33 nM of IgG1 control Ab, m15-IgG1, PF-06747143, or rituximab.

Figure 3.

Efficacy of PF-06747143 and role of Fc-effector function in the Ramos NHL efficacy model. Ramos cells were implanted subcutaneously (5 × 106 cells), and when mean tumor volume reached 350 to 400 mm3, mice were randomized (n = 10-12 per group). Arrows indicate Ab treatment days. Data points represent the mean tumor volume ± SEM. (A) Animals were treated weekly, subcutaneously, with Ab’s at 10 mg/kg for 2 doses, on days 1 and 8. (B) Animals were treated weekly, subcutaneously, with control IgG1 Ab at 10 mg/kg and PF-06747143 at 0.1, 1, 10, and 30 mg/kg for 4 doses, or until day 21. (C) Animals were treated weekly, subcutaneously, with m15-IgG1 or m15-IgG4 Ab’s at 1 and 10 mg/kg for 3 doses. Study was terminated at day 15. (D) Antibody exposure was evaluated 24 hours after last dose. Serum from 3 animals per group was collected, and human IgG concentration determined by enzyme-linked immunosorbent assay. Bars points represent the mean concentration ± SEM. (E) Cleaved caspase-3 in tumor tissue collected 48 and 72 hours post–Ab treatment at 10 mg/kg was determined by western blot (n = 3 animals per group).

Figure 4.

PF-06747143 greatly reduces BM tumor burden as a monotherapy and in combination with SOCs in a disseminated MM model. OPM-2-Luc MM cells were implanted IV (5 × 106 cells) and allowed to spontaneously migrate and home in the BM for 8 days, when animals were randomized based on luciferase activity detected in the large bones (n = 10 per group). Animals showing hind leg paralysis were euthanized, and this was the survival end point of the study. (A) Treatment schematic representation. Animals were treated with 10 mg/kg of IgG1 control and PF-06747143 Ab’s, subcutaneously, weekly, for 5 doses. Melphalan was dosed 1 mg/kg, intraperitoneally (IP), twice a week, for a total of 4 cycles. (B) Tumor burden was determined by bioluminescence imaging and quantification. Data points represent the mean bioluminescence ± SEM. (C) Whole body bioluminescence representative imaging showing tumor burden in n = 5 mice per group over time. (D) Kaplan-Meier survival curve. (E) Treatment schematic representation. Animals were treated with 1 mg/kg of IgG1 control and PF-06747143 Ab’s, subcutaneously, weekly, for a total of 7 doses. Bortezomib was dosed at 0.5 mg/kg, administered IP, 2 times per week, for a total of 4 cycles. (F) Tumor burden was determined by bioluminescence imaging quantification. Data points represent the mean bioluminescence ± SEM. (G) Whole body bioluminescence representative images showing tumor burden in n = 5 mice per group over time. (H) Kaplan-Meier survival curve.

Figure 5.

PF-06747143 reduces BM tumor burden in a dose-response dependent manner in a disseminated AML tumor model. MV4-11-Luc AML cells were implanted IV (1 × 106 cells) and allowed to spontaneously migrate and home in the BM for 11 days, when animals were randomized (n = 5-10 per group). (A) Treatment schematic representation. Animals were treated with IgG1 control or PF-06747143 Ab’s, subcutaneously, weekly, for 4 doses. Daunorubicin was dosed at 2 mg/kg, IV, 3 times (days 1, 3, and 5). Crenolanib was dosed at 7.5 mg/kg, IP, twice a day, on days 11 through 15 and days 25 through 29. (B) Tumor burden was determined by bioluminescence imaging and quantification. Data points represent the mean bioluminescence ± SEM. (C) Whole body bioluminescence representative imaging showing tumor burden in n = 5 mice per group over time. (D) On day 35, 3 days following the final Ab treatment, tumor burden was evaluated in PB and BM cells by flow cytometry, using hCD45 and hCD33 Ab’s as AML markers (n = 5-10 mice per group). Data points represent each individual mouse. (E) Kaplan-Meier survival curve (n = 5 animals per group), using hind leg paralysis as the end point.

Figure 6.

PF-06747143 reduces BM tumor burden in a PDX chemoresistant AML tumor model. (A) NSG mice received 150 cGy whole body irradiation, and within 24 hours, 1.5 × 106 PDX BM cells were implanted IV and allowed to spontaneously migrate and home in the BM for 43 days, when tumor burden in PB reached 0.8% to 1.6%. Animals were then randomized in 5 animals per treatment group and treated with IgG1 control or PF-06747143 Ab’s, subcutaneously, weekly, for 8 doses. Daunorubicin was dosed at 1.5 mg/kg, IV, 3 times per week, for 2 cycles. Ara-C was dosed at 15 mg/kg, IP, once per day, from days 43 through 50 and days 64 through 71. On day 99, n = 5 animals per group were euthanized, and PB and BM samples were analyzed by flow cytometry to detect CXCR4+ malignant AML cells (hCD45+ and hCD33+) (B) or total AML cells (hCD45+ and hCD33+) (C).

Figure 7.

Role of Fc-Effector function in cell mobilization and normal hematopoiesis. (A) Cell mobilization into the PB was detected by flow cytometry, using hCD45 and hCD33 Ab’s following a single dose of PF-06747143 (10 mg/kg) in MV4-11 AML tumor-bearing mice. Data represent mean hCD45+ and hCD33+ staining in PB relative to baseline (time 0 hours) ± SEM. (B) Cynomolgus monkeys (n = 3 per group) received a single dose of m15-IgG1 or m15-IgG4, at 50 mg/kg, or vehicle (control). Mean absolute PB WBC numbers ± SEM are shown over time. (C) Cynomolgus monkeys (n = 2 per group) received 2 weekly doses of PF-06747143 (10 mg/kg) or vehicle, at 0 and 168 hours. Mean absolute PB WBC numbers ± SEM are shown over time. (D) Cynomolgus monkeys (n = 8-12 per group) received 2 weekly doses of PF-06747143 (10 mg/kg), at 0 and 168 hours. PB number of hematopoietic progenitor CD34+ cells ± SEM, relative to each individual baseline level, are shown over time.