CD38 is a type II transmembrane glycoprotein with multiple functions and expressed lower on normal blood cells and higher on hematologic tumor cells [1, 2]. Anti-CD38 mAbs were effective in some hematological malignancies, especially multiple myeloma (MM) [3]. Although both daratumumab and isatuximab have significantly improved the outcome of patients with MM, incomplete responses and on-target/off-tumor effects emerge during the treatment [4, 5]. Herein, we described FTL004, a novel humanized IgG1κ anti-CD38 mAb possessing novel properties.
FTL004 exhibited similar affinities to CD38 (KD of 2.55, 3.84, and 1.2 nM, respectively) in surface plasmon resonance (Fig. 1a) and to CD38-positive cell lines in flow cytometry (Fig. 1b), compared with daratumumab or isatuximab. However, these antibodies bound differently to blood cells. Daratumumab or isatuximab bound to RBCs to different extents from healthy donors, while the binding of FTL004 to RBCs was virtually undetectable (Fig. 1c). This property should result in a more favorable pharmacokinetic of FTL004 by circumventing binding to CD38 on circulating RBCs [6] and a better safety profile by minimizing side effects associated with RBC binding. Moreover, FTL004 did not agglutinate RBCs in indirect antiglobulin tests (Fig. 1d), which could avoid the interferences with blood transfusion that are observed in other anti-CD38 mAbs [7, 8].
For other immune cells, FTL004 bound to most subpopulation cells of peripheral blood mononuclear cells (PBMCs) in a comparable intensity with daratumumab or isatuximab (Fig. 1e). Unexpectedly, FTL004 exhibited a slightly higher binding to CD8+ T cells and a much higher binding to NK cells (Fig. 1f). These events reflected that the epitope of FTL004 binding on CD38 may be distinct. Although anti-CD38 mAbs require a high threshold for CD38 expression to induce cell death, we must be wary of its possible off-target effects. PBMCs coculture assays showed that there was no depletion in T and B cells, indicating the limited off-tumor toxicity of FTL004 (Fig. 1g). But as expected, NK cells, with higher expression of CD38, were reduced to an acceptable level [9].
Then, the CD38/FTL004 complex structure was determined using Cryo-electron microscopy at 3.86 Å resolution (Fig. 1h, i). Of note, there seem to be two epitope centers of FTL004, one of them includes from Pro52 to His56 and the other one is 91TQTV94. Superimposition of the CD38/daratumumab (PDB code 7DHA) complex and CD38/isatuximab (PDB code 4CMH) complex onto the CD38/FTL004 complex revealed that the epitopes of FTL004 share no common residue of CD38 with daratumumab, whereas one of the epitopes (91TQTV94) of FTL004 partly overlaps with that of isatuximab (Fig. 1j).
Next, to evaluate the tumor-killing capacity of FTL004, pro-apoptotic activity was first analyzed. We found that FTL004 had a superior ability to induce direct apoptosis independent of cross-linking reagents against CD38-positive cell lines (Fig. 2a) and primary MM cells (Fig. 2b) than isatuximab. Particularly, the highest apoptosis rate of FTL004 on primary MM cells was detected to be up to 60%. On the other hand, the Fc domain of FTL004 was engineered (S240D/I333E mutation) to elevate the affinity to human Fcγ receptors, leading to the enhancement of antibody-dependent cellular cytotoxicity (ADCC) [10]. In ADCC bioassays with Jurkat-Lucia-CD16a-NFAT cells [11], FTL004 showed a distinctly higher intensity of ADCC, with EC50 values of 2–8 ng/mL, compared with daratumumab and isatuximab (Fig. 2c). Consistent results were obtained in primary MM cells (Fig. 2d).
To investigate further, we examined the ADCC of FTL004 with healthy PBMCs. PBMCs exhibited enhanced ADCC activity against CHO-CD38+ cells in the presence of FTL004, with threefold higher than daratumumab (Fig. 2e). Meanwhile, FTL004 induced stronger ADCP with 74% phagocytosed CHO-CD38+ cells in an EC50 value of 40 ng/mL (Fig. 2f). However, FTL004 induced a lower CDC than daratumumab of CHO-CD38+ cells (Fig. 2g). In vivo studies were further carried out, and FTL004 was examined in Ramos, H929, and MM1S-bearing NOD-SCID mice (Fig. 2h). FTL004 treatment resulted in significant inhibition of tumor growth compared to PBS treatment.
In summary, FTL004 is a novel CD38 mAb that owns unique properties. Although the mechanism by which FTL004 binds differently to tumor cells versus RBCs remains unclear, it is expected that FTL004 owns limited on-target/off-tumor effects. The results above suggest that FTL004 is a therapeutic Ab with high potential for the treatment of MM and non-Hodgkin lymphoma.