Anti-tumor activity and toxicokinetics analysis of MGAH22, an anti-HER2 monoclonal antibody with enhanced Fcγ receptor binding properties

Abstract

Introduction: Response to trastuzumab in metastatic breast cancer correlates with expression of the high binding variant (158V) of the activating Fcγ receptor IIIA (CD16A). We engineered MGAH22, a chimeric anti-HER2 monoclonal antibody with specificity and affinity similar to trastuzumab, with an Fc domain engineered for increased binding to both alleles of human CD16A.

Methods: MGAH22 was compared to an identical anti-HER2 mAb except for a wild type Fc domain. Antibody-dependent cell cytotoxicity (ADCC) assays were performed with HER2-expressing cancer cells as targets and human PBMC or purified NK cells as effectors. Xenograft studies were conducted in mice with wild type murine FcγRs; in mice lacking murine CD16; or in mice lacking murine CD16 but transgenic for human CD16A-158F, the low-binding variant. The latter model reproduces the differential binding between wild type and the Fc-optimized mAb for human CD16A. The JIMT-1 human breast tumor line, derived from a patient that progressed on trastuzumab therapy, was used in these studies. Single and repeat dose toxicology studies with MGAH22 administered intravenously at high dose were conducted in cynomolgus monkeys.

Results: The optimized Fc domain confers enhanced ADCC against all HER2-positive tumor cells tested, including cells resistant to trastuzumab's anti-proliferative activity or expressing low HER2 levels. The greatest improvement occurs with effector cells isolated from donors homozygous or heterozygous for CD16A-158F, the low-binding allele. MGAH22 demonstrates increased activity against HER2-expressing tumors in mice transgenic for human CD16A-158F. In single and repeat-dose toxicology studies in cynomolgus monkeys, a species with a HER2 expression pattern comparable to that in humans and Fcγ receptors that exhibit enhanced binding to the optimized Fc domain, MGAH22 was well tolerated at all doses tested (15-150 mg/kg) and exhibited pharmacokinetic parameters similar to that of other anti-HER2 antibodies. Induction of cytokine release by MGAH22 in vivo or in vitro was similar to that induced by the corresponding wild type mAb or trastuzumab.

Conclusions: The data support the clinical development of MGAH22, which may have utility in patients with low HER2 expressing tumors or carrying the CD16A low-binding allele.

Figure 1

HER2-binding and anti-proliferative activity. (a) HER2-binding activity of MGAH22 was compared with RES120 or trastuzumab by antigen-capture enzyme-linked immunosorbent assay. EC₅₀ (and 95% confidence interval) values were 39.33 (29.45 to 52.52) ng/mL for MGAH22 and 45.75 (33.37 to 62.67) ng/mL for RES120 (left panel) and 28.76 (24.96 to 33.15) for MGAH22 and 27.28 (23.6 to 31.53) ng/mL for trastuzumab (right panel). (b) Proliferation of JIMT-1 and SKBR-3 cells in the presence of MGAH22, RES120, or trastuzumab. Data are presented as mean ± standard error of the mean. EC₅₀, effective concentration for 50% binding; MGAH22, chimeric anti-HER2 monoclonal antibody with an optimized Fc domain; Neg ctrl mAb, negative control monoclonal antibody; RES120, chimeric anti-HER2 monoclonal antibody with wild-type human immunoglobulin G 1 Fc domain; RLU, relative light units.

Figure 2

Binding of monoclonal antibodies with wild-type or optimized Fc domains to FcγRs. Representative surface plasmon resonance traces for binding of fixed concentrations of soluble human FcγRs or C1q (a), cynomolgus monkey FcγRs (b), or murine FcγRs (c) to ch4D5 (contains wild-type-Fc domain) or ch4D5-0264 (contains MGFc0264) captured on immobilized recombinant human HER2 protein are shown. hCD16A-158V, hCD16A-158F, and hCD64 were analyzed as soluble monomeric extracellular domains (ECDs), whereas hCD32A-131R, hCD32A-131H, and hCD32B were analyzed as soluble dimeric extracellular domain Fc-fusions (Fc_N297Q or Fc_D265A). Values of the equilibrium dissociation constant (K_D) from full-range titration studies were determined by the fitting of equilibrium responses to a steady-state affinity model for hCD16 and hCD32 receptors or by a global fit to 1:1 binding model for hCD64 interactions that did not reach a steady state. FcγR, Fc-gamma receptor; RU, relative units.

Figure 3

In vitro antibody-dependent cell-mediated cytotoxicity (ADCC) activity. (a-e) ADCC with JIMT-1 as target cells and purified natural killer (NK) cells from two or three independent donors for each CD16A genotype (F/F, F/V, or V/V) as effectors using a 3:1 effector/target ratio. (f-m) ADCC with breast and non-breast cancer cell lines expressing HER2 at different levels as target cells and peripheral blood mononuclear cells from three to six independent CD16A-158F carriers as effectors using a 30:1 effector/target ratio. Fold increases in percentage maximal lysis by MGAH22 relative to RES120 and EC₅₀ values are plotted according to CD16A genotype of effectors (d, e) or number of HER2-binding sites per target cell (l, m). Data are presented as mean ± standard error of the mean. EC₅₀, effective concentration for 50% lysis; MGAH22, chimeric anti-HER2 monoclonal antibody with an optimized Fc domain; RES120, chimeric anti-HER2 monoclonal antibody with wild-type human immunoglobulin G 1 Fc domain.

Figure 4

In vivo efficacy. (a) Wild-type FcγR mice (10 per group): JIMT-1 cells implanted subcutaneously (s.c.) and monoclonal antibodies (mAbs) at 4 mg/kg administered five times at weekly intervals beginning at day 0. The first day of significant reduction in tumor size occurred at day 30 for both MGAH22 (P < 0.01) and RES120 (P < 0.01) compared with phosphate-buffered saline (PBS). (b) mCD16^-/- mice (11 per group): JIMT-1 cells implanted s.c. and mAbs at 2 mg/kg administered six times at weekly intervals beginning at day 0. (c) mCD16^-/- hCD16A⁺ mice (8 per group): JIMT-1 cells implanted s.c. and mAbs at 2 mg/kg administered six times at weekly intervals beginning at day 0. The first day of significant reduction in tumor sizes occurred at day 37 for MGAH22 compared with PBS (P < 0.001), at day 44 for RES120 compared with PBS (P < 0.01), and at day 61 for MGAH22 compared with RES120 (P < 0.05). (d, e) mCD16^-/- hCD16A⁺ mice (10 per group): JIMT-1 cells implanted s.c. and tumors of approximately 200 mm³ allowed to form; RES120 (d) or MGAH22 (e) administered six times at weekly intervals beginning at day 6 at 0.01, 0.1, 1, or 10 mg/kg. The first day of significant reduction in tumor size occurred at day 32 for 1 or 10 mg/kg MGAH22 compared with PBS (P < 0.01), at day 41 for 10 mg/kg RES120 compared with PBS, at day 47 for 1 mg/kg MGAH22 compared with 1 mg/kg RES120 (P < 0.05), and at day 63 for 10 mg/kg MGAH22 compared with 10 mg/kg RES120 (P < 0.01). Data are presented as mean ± standard error of the mean. The first days of significant reduction in tumor sizes are indicated for MGAH22 or RES120 compared with vehicle (*) and for MGAH22 compared with RES120 (#). MGAH22, chimeric anti-HER2 monoclonal antibody with an optimized Fc domain; RES120, chimeric anti-HER2 monoclonal antibody with wild-type human immunoglobulin G 1 Fc domain.

Figure 5

Pharmacokinetics in cynomolgus monkeys. Serum concentration-versus-time profiles for intravenous administration of MGAH22 or RES120 at a single 50 mg/kg dose (a) or MGAH22 at 15 (b), 50 (c), or 150 (d) mg/kg weekly for 6 weeks. Data are presented as mean ± standard error of the mean. MGAH22, chimeric anti-HER2 monoclonal antibody with an optimized Fc domain; RES120, chimeric anti-HER2 monoclonal antibody with wild-type human immunoglobulin G 1 Fc domain.

Figure 6

MGAH22-induced IL-6 release in vivo and in vitro. (a) Serum IL-6 levels in single- and repeat-dose studies in cynomolgus monkeys. When MGAH22-treated groups were compared with RES120-treated groups (single-dose study) or with vehicle control group (repeat-dose study), there were no statistically significant changes in IL-6 levels. (b) In vitro IL-6 release from human peripheral blood mononuclear cells (PBMCs) incubated with the indicated antibodies on uncoated plates or plates coated with recombinant HER2 antigen. Statistically significant changes in IL-6 levels (P = 0.0313) were noted for MGAH22 compared with RES120 at concentrations of 1 or 10 μg/mL in the presence of immobilized HER2, but there were no significant differences between MGAH22 and trastuzumab. Data are presented as mean ± standard error of the mean. IL-6, interleukin-6; MGAH22, chimeric anti-HER2 monoclonal antibody with an optimized Fc domain; RES120, chimeric anti-HER2 monoclonal antibody with wild-type human immunoglobulin G 1 Fc domain.