FTL004, an anti-CD38 mAb with negligible RBC binding and enhanced pro-apoptotic activity, is a novel candidate for treatments of multiple myeloma and non-Hodgkin lymphoma

Abstract

Anti-CD38 monoclonal antibodies (mAbs), daratumumab, and isatuximab have represented a breakthrough in the treatment of multiple myeloma (MM). Recently, CD38-based mAbs were expected to achieve increasing potential beyond MM, which encouraged us to develop new anti-CD38 mAbs to meet clinical needs. In this study, we developed a novel humanized anti-CD38 antibody, FTL004, which exhibited enhanced pro-apoptotic ability and negligible binding to red blood cells (RBCs). FTL004 presented a better ability to induce direct apoptosis independent of Fc-mediated cross-linking against lymphoma and MM cell lines as well as primary myeloma cells derived from MM patients. For instance, FTL004 induced RPMI 8226 cells with 55% early apoptosis cells compared with 20% in the isatuximab-treated group. Of interest, FTL004 showed ignorable binding to CD38 on human RBCs in contrast to tumor cells, even at concentrations up to 30 μg/mL. Furthermore, with an engineered Fc domain, FTL004 displayed stronger antibody-dependent cellular cytotoxicity (ADCC) against CD38+ malignant cells. In vivo MM and non-Hodgkin lymphoma tumor xenograft models showed that FTL004 possessed an effective anti-tumor effect. Cryo-electron microscopy structure resolved two epitope centers of FTL004 on CD38: one of which was unique while the other partly overlapped with that of isatuximab. Taken together, FTL004 distinguishes it from other CD38 targeting mAbs and represents a potential candidate for the treatment of MM and non-Hodgkin lymphoma.

Keywords: CD38; Direct apoptosis; Monoclonal antibodies; Multiple myeloma; Red blood cells.

Fig. 1

Distinct binding profile of FTL004 to CD38+ tumor cells, RBCs, and immune cells. a Affinity analysis of FTL004 to human CD38 as measured by Biacore 8 K (KD = 2.55 × 10^–9 mol/L). b EC50 values for anti-CD38 mAbs binding to diverse cells. DARA, daratumumab; ISA, isatuximab. c Binding of FTL004, daratumumab or isatuximab to freshly isolated RBCs from healthy donors. d Indirect antiglobulin tests for CD38 mAbs to the same three healthy donors RBCs above. e The percentage of binding of CD38 mAbs to normal PBMCs (n = 4), including to CD45+ CD3+ T cells, CD3+ CD4+ T cells, CD3+ CD8+ T cells, CD45+ CD19+ B cells and CD3-CD56+ NK cells. Data show means ± SD and unpaired Student’s t tests were used to determine the statistical significance between daratumumab versus FTL004. *P < 0.05, **P < 0.01 and ***P < 0.001. f Flow cytometry dot plots showing the binding of anti-CD38 mAbs to NK cells and CD8+ T cells. g PBMCs from normal donors (n = 6) were pretreated with 1 μg/mL FTL004 or isatuximab for 3 days. Flow cytometry was used to determine the percentage of CD3+ T cells, CD4+ T cells, CD8+ T cells, B cells, and NK cells in lymphocytes. Data were then normalized to isotype controls and fold changes (means ± SD) are shown. Isotype cells were used as controls in settings. h The overall structure of the CD38/FTL004 Fab complex. CD38 and the heavy and light chains of FTL004 are colored gray, purple, and green, respectively. i Stereoscopic view of the direct hydrogen bonds, salt bridge, in the interface between CD38 and FTL004. j Comparison of the binding of FTL004, daratumumab, and isatuximab. Surface representation of the epitopes of FTL004 (green), daratumumab (blue), isatuximab (red) and superposition part of FTL004 and isatuximab (yellow) on CD38 (gray)

Fig. 2

Anti-tumor efficacy of FTL004. a Induction of apoptosis by FTL004, daratumumab, or isatuximab in various cell lines, cells were treated with anti-CD38 mAbs 1.5 μg/mL for 24 h. Data represent means of triplicates and SD. Statistical significance between FTL004 and hIgG groups was determined by unpaired Student’s t tests. *P < 0.05; **P < 0.01, ***P < 0.001. b Apoptosis induction by FTL004 in primary MM cells. c, d ADCC reporter bioassays to various CD38+ tumor cell lines (c) and MM primary cells (d) of FTL004, daratumumab, or isatuximab. Data represent means of triplicates and SD. e–g Representative dose–response curve examples for ADCC activity (e), ADCP activity (f), and CDC activity (g) of FTL004 or daratumumab to CHO-CD38+ cells. Data show mean ± SD of three experiments. h Anti-tumor efficacy of FTL004 in lymphoma cell Ramos, MM cell H929, MM1S xenograft models. NOD-SCID mice bearing xenografts were treated with administration of PBS, daratumumab, or FTL004. Tumor burdens were monitored. Each point on the graph represents the average tumor volume