ARGX-110, a highly potent antibody targeting CD70, eliminates tumors via both enhanced ADCC and immune checkpoint blockade

Abstract

Overexpression of CD70 has been documented in a variety of solid and hematological tumors, where it is thought to play a role in tumor proliferation and evasion of immune surveillance. Here, we describe ARGX-110, a defucosylated IgG1 monoclonal antibody (mAb) that selectively targets and neutralizes CD70, the ligand of CD27. ARGX-110 was generated by immunization of outbred llamas. The antibody was germlined to 95% human identity, and its anti-tumor efficacy was tested in several in vitro assays. ARGX-110 binds CD70 with picomolar affinity. In depletion studies, ARGX-110 lyses tumor cells with greater efficacy than its fucosylated version. In addition, ARGX-110 demonstrates strong complement-dependent cytotoxicity and antibody-dependent cellular phagocytosis activity. ARGX-110 inhibits signaling of CD27, which results in blocking of the activation and proliferation of Tregs. In a Raji xenograft model, administration of the fucosylated version of ARGX-110 resulted in a prolonged survival at doses of 0.1 mg/kg and above. The pharmacokinetics of ARGX-110 was tested in cynomolgus monkeys; the calculated half-life is 12 days. In conclusion, ARGX-110 is a potent blocking mAb with a dual mode of action against both CD70-bearing tumor cells and CD70-dependent Tregs. This antibody is now in a Phase 1 study in patients with advanced malignancies expressing CD70 (NCT01813539).

Keywords: ARGX-110; CD70; POTELLIGENT®; immune checkpoint blockade.

Figures

Figure 1. Epitope mapping was done by competition experiments using the 786-O cell line. CD70 was saturated first with the Fabs and binding of biotinylated anti-CD70 mAbs was tested in FACS. Blockage of mAb binding indicates that the Fab and mAb share the same epitope. The epitopes of 9D1, 9B2, 5F4, 5B2, 9G2 and 4D2 overlap, whereas the epitope of 1C2, 9E1 and 7H8 are different. The Fabs recognize at least three different epitopes.

Figure 2. Binding and blocking activity of ARGX-110 on U266 (multiple myeloma), Granta519 and Mino-1 (Mantle cell lymphoma) and Raji (Burkitt lymphoma) cell lines. (A) binding of varying concentrations of ARGX-110 to CD70-positive cell lines and the CD70 negative cell line SUP-T1 as determined by flow cytometry. Data presented as mean fluorescent intensity (MFI) of duplicate measurements and SD (B) blocking of CD27 signaling by ARGX-110. CD70-positive cell lines, HEK293 cells with or without transfection with CD70 and crosslinked recombinant CD70 were co-cultured with HT1080-CD27-positive cells in the presence of varying concentrations of ARGX-110. IL-8 was measured by ELISA as a marker for CD27 signaling. Data presented as mean of triplicate measurements and SD.

Figure 3. Effector functions of ARGX-110. (A) ADCC activity measured with Cr51 release assay on 786-O cells (RCC). ARGX-110 is associated with enhanced ADCC activity compared with the fucosylated version of the antibody, 41D12. (B) De-fucosylation does not impair CDC on U266 cells (multiple myeloma) nor (C) ADCP activity of ARGX-110 (on 786-O cells). All data are presented as mean of triplicate measurements and SD.

Figure 4. Depletion studies on cell lines representing different histologies. (A-B) Cell lines were admixed with freshly isolated PBMCs from healthy donors and ARGX-110 or 41D12 was added at 5 µg/ml. The samples were incubated for 2 d and analyzed by FACS for the lysis of the cell lines by looking at specific cell line markers. All data are presented as mean of duplicate measurements. (C) % lysis is plotted against CD70 signal in FACS using anti-CD70-PE demonstrating that depletion is independent of CD70 copy number.

Figure 5. Blocking of Treg activity by ARGX-110 demonstrated in an in vitro model. (A) PBMCs from a healthy donor were admixed with CD70-positive tumor cell lines (SUDHL6, Raji and U266) in the presence of soluble anti-CD3 mAb and ARGX-110. ARGX-110 lysed the cell lines during the experiment. (B) ARGX-110 blocks Treg proliferation. More Tregs are formed in the wells where the CD70-positive cell lines were added than in the wells without addition of cell lines and this can be blocked with ARGX-110. Results are shown as mean and SD of duplicates. (C) Proliferation of the Tregs is dependent on CD27 signaling and can be blocked with ARGX-110. After two days of incubation, ARGX-110 reduces sCD27 (as determined by ELISA) levels further witnessing of a role for CD70 in Treg proliferation.

Figure 6. In vivo efficacy study using Raji xenograft model. (A) Kaplan-Meier plot showing the antitumor effect of 41D12 in a disseminated Burkitt lymphoma xenograft model. SCID mice were injected intravenously with 106 Raji B lymphoma cells, then treated with 41D12 or isotype control at the doses indicated. Mice were treated two times per week thereafter and received a total of five doses (n = 9 mice per group). (B) A plasma sample was obtained for each mouse at the time of sacrifice. sCD27 levels were determined by ELISA. Tumor bearing mice (n = 28) had higher levels of sCD27 than non-tumor bearing mice (n = 42). Statistics was done comparing groups treated with 41D12 vs. the isotype control.