Preclinical characterization of bemarituzumab, an anti-FGFR2b antibody for the treatment of cancer

Abstract

Bemarituzumab (FPA144) is a first-in-class, humanized, afucosylated immunoglobulin G1 monoclonal antibody (mAb) directed against fibroblast growth factor receptor 2b (FGFR2b) with two mechanisms of action against FGFR2b-overexpressing tumors: inhibition of FGFR2b signaling and enhanced antibody-dependent cell-mediated cytotoxicity (ADCC). Bemarituzumab is being developed as a cancer therapeutic, and we summarize here the key nonclinical data that supported moving it into clinical trials. Bemarituzumab displayed sub-nanomolar cross-species affinity for FGFR2b receptors, with >20-fold enhanced binding affinity to human Fc gamma receptor IIIa compared with the fucosylated version. In vitro, bemarituzumab induced potent ADCC against FGFR2b-expressing tumor cells, and inhibited FGFR2 phosphorylation and proliferation of SNU-16 gastric cancer cells in a concentration-dependent manner. In vivo, bemarituzumab inhibited tumor growth through inhibition of the FGFR2b pathway and/or ADCC in mouse models. Bemarituzumab demonstrated enhanced anti-tumor activity in combination with chemotherapy, and due to bemarituzumab-induced natural killer cell-dependent increase in programmed death-ligand 1, also resulted in enhanced anti-tumor activity when combined with an anti-programmed death-1 antibody. Repeat-dose toxicity studies established the highest non-severely-toxic dose at 1 and 100 mg/kg in rats and cynomolgus monkeys, respectively. In pharmacokinetic (PK) studies, bemarituzumab exposure increase was greater than dose-proportional, with the linear clearance in the expected dose range for a mAb. The PK data in cynomolgus monkeys were used to project bemarituzumab linear PK in humans, which were consistent with the observed human Phase 1 data. These key nonclinical studies facilitated the successful advancement of bemarituzumab into the clinic.

Keywords: Bemarituzumab; afucosylated antibody; antibody-dependent cell-mediated cytoxicity; phosphorylation in vitro; anti-FGFR2b antibody; anti-tumor efficacy; cell proliferation in vitro; fibroblast growth factor receptor; pharmacokinetics; toxicology.

Figure 1.

Bemarituzumab inhibits FGFR2 phosphorylation and FGFR2b-mediated proliferation of SNU-16 cells. a: FGFR2 phosphorylation (pFGFR2) in SNU-16 cells treated with varying concentrations of bemarituzumab followed by 100 ng/mL FGF7 is shown. FGFR2 phosphorylation was measured via ELISA. b: Proliferation of SNU-16 cells treated with varying concentrations of bemarituzumab followed by 100 ng/mL FGF7 is shown. Proliferation was measured via cell viability assay. The experiments were performed in triplicate; data are presented as mean ±standard error of mean (SEM) relative light units (RLU)

Figure 2.

Bemarituzumab elicits potent induction of ADCC activity against FGFR2b-expressing cells. Specific lysis of target cells as a percentage of maximal lysis in the presence of varying concentrations of bemarituzumab or FPA144-F is shown: Ba/F3 cells expressing full-length human FGFR2b treated with bemarituzumab (Bema/FGFR2b); Ba/F3 cells expressing full-length human FGFR2b treated with FPA144-F (FPA144-F/GFGFR2b); Ba/F3 cells expressing full-length human FGFR2c treated with bemarituzumab (Bema/FGFR2c). Data are presented as mean ±standard error of mean (SEM); n = 3 per concentration/group

Figure 3.

Bemarituzumab inhibits tumor growth in OCUM-2M tumor-bearing mice. Tumor volume (mm³) over time in OCUM-2M tumor-bearing mice treated with twice per week IV bemarituzumab at varying dose levels is shown. Albumin (5 mg/kg, IV) was used as the negative control. The first day of the treatment was day 4 post tumor implantation. Data are presented as mean ±standard error of mean (SEM); n = 15 per group

Figure 4.

Bemarituzumab decreases FGFR2 signaling in SNU-16 tumor-bearing mice. Western blot analyses of pan-phosphorylated FGFR (phospho-FGFR), total FGFR2b, phosphorylated FRS2 (phospho-FRS2), and total FRS2 in tumors from SNU-16 tumor-bearing mice are shown. All results were compared to a common housekeeping enzyme (GAPDH). a: Tumors from mice treated with a single IV dose of bemarituzumab (10 mg/kg) were collected at 2, 6, 24, and 72 hours post treatment (n = 3 per time point). Control groups included tumors collected at 2 hours from naive animals (n = 2) and at 24 hours post IgG treatment (10 mg/kg; n = 3). Bemarituzumab decreased phospho-FGFR and phospho-FRS2 within 2 hours, and decreased total FGFR2b levels within 6 hours. b: Tumors from mice treated with twice weekly IP doses of 10 mg/kg bemarituzumab or albumin were collected at 82 days post first bemarituzumab/albumin treatment (n = 3 per group). Compared with the albumin control group, bemarituzumab decreased phospho-FGFR, phospho-FRS2, and total FGFR2b levels

Figure 5.

Bemarituzumab, but not an ADCC-deficient FGFR2b antibody, leads to tumor suppression in a syngeneic tumor model with modest FGFR2b expression. a: Tumor volume (mm³) over time in mice bearing 4T1 mammary orthotopic tumors treated with twice per week bemarituzumab (10 mg/kg, IP) or bemarituzumab-N297Q (10 mg/kg, IP) are shown. Human Fc-IgG1 (hFc-G1,10 mg/kg, IP) was used as the negative control. The first day of treatment was Day 11 post tumor implantation. Data are presented as mean ±standard error of mean (SEM); n = 15 per group. b: at day 25 post tumor implantation, mean tumor volumes from mice in panel a were significantly different (****p < .0001). Statistical significance was determined by 1 way ANOVA followed by Tukey’s multiple comparisons test. Each symbol represents an individual animal

Figure 6.

Bemarituzumab induces tumor-specific innate and adaptive immune profile changes. Mice bearing 4T1 tumors were treated with 20 mg/kg IP bemarituzumab (Bema), bemarituzumab-N297Q (Bema-N297Q), or human Fc-IgG1 (hFc-G1) on day 0 (when tumors reached approximately 150 mm³) and day 3, and then euthanized 24 hours later on day 4 for immunohistochemistry analysis. Representative fluorescence of NKp46, PD-L1, and CD3-positive T cells (CD3 T cells) 24 hours post second dose of treatment are shown

Figure 7.

Bemarituzumab-induced increases in PD-L1 and CD3-positive T cells depends on NK cell recruitment. Mice bearing 4T1 tumors were treated with either: human Fc-IgG1 negative control (10 mg/kg, IP) on day 0 (when tumors reached approximately 125 mm³) and day 3 (hFc-G1), anti-ASGM1 (50 mg/kg, IV) once on day 0, bemarituzumab (10 mg/kg, IP) on day 0 and day 3 (Bema), or anti-ASGM1 (50 mg/kg, IV) on day 0 and bemarituzumab (10 mg/kg, IP) on day 0 and day 3 (anti-ASGM1 + Bema). Mice were euthanized on day 4 for immunohistochemistry analysis. Representative fluorescence of NKp46, PD-L1, and CD3-positive T cells (CD3⁺ T cells) are shown

Figure 8.

Bemarituzumab displays enhanced anti-tumor activity when combined with anti-PD-1 or chemotherapeutics. a: Tumor volume (mm³) over time in mice bearing 4T1 mammary orthotopic tumors treated with twice per week bemarituzumab (10 mg/kg, IP), anti-PD-1 (5 mg/kg, IP), or bemarituzumab (10 mg/kg, IP) and anti-PD-1 (5 mg/kg, IP) in combination (Combo) are shown. Albumin (10 mg/kg, IP) was used as the negative control. The first day of the treatment was day 12 post tumor implantation. Data are presented as mean ±standard error of mean (SEM); n = 15 per group. b: at day 19 post tumor implantation, mean tumor volumes from mice in panel a were either statistically significant (**p < .01, ***p < .001) or not statistically significant (ns). Each symbol represents an individual animal. c: Tumor volume (mm³) over time in OCUM-2 M tumor-bearing mice treated with twice per week bemarituzumab (5 mg/kg, IP), weekly 5-FU (30 mg/kg, IP) and oxaliplatin (5 mg/kg, IP), or twice per week bemarituzumab (5 mg/kg, IP) and weekly 5-FU (30 mg/kg, IP)/oxaliplatin (5 mg/kg, IP) in combination (Combo) are shown. Human IgG1(5 mg/kg, IP) was used as the negative control (hIgG1). The first day of the treatment was post tumor implantation day 5 for 5-FU/oxaliplatin and day 11 for bemarituzumab. Data are presented as mean ±SEM; n = 9–10 per group. d: at day 63 post tumor implantation, mean tumor volumes from mice in Panel c were either statistically significant (*p < .05, ***p < .001, ****p < .0001) or not statistically significant (ns). Each symbol represents an individual animal. Statistical significance was determined by 1 way ANOVA followed by Tukey’s multiple comparisons

Figure 9.

Bemarituzumab demonstrates biphasic disposition in rats and cynomolgus monkeys following a single IV injection. a: Bemarituzumab plasma concentration in rats following a single IV injection at 1.5 mg/kg (●; n = 4), 10 mg/kg (■; n = 4), or 30 mg/kg (▲; n = 3). Based on the concentration profiles, 2 animals (1 in the 1.5 mg/kg group and 1 in the 10 mg/kg group) were suspected of inadvertent subcutaneous dosing. b: Plasma concentrations of bemarituzumab and FPA144-F in cynomolgus monkeys following a single IV injection of bemarituzumab 10 mg/kg (–■̶; n = 1), bemarituzumab 13.5 mg/kg (–●̶; n = 1), or FPA144-F 10 mg/kg (-●-, -■-; n = 2). Each symbol represents a concentration time point for an individual animal. The lower limit of quantification (–) was 50 ng/mL

Figure 10.

Projected human plasma concentration using PK data from cynomolgus monkeys. Predicted bemarituzumab plasma concentration-time profile (median [solid line], 5% and 95% quantiles [dashed lines], 90% prediction interval [shaded area]) in humans following 1 mg/kg IV administration. Bemarituzumab concentration-time profiles were scaled from cynomolgus monkey PK data using Dedrick approach with exponent of 0.85 and 1 for clearance and V₁, respectively