CLN-619, a MICA/B monoclonal antibody that promotes innate immune cell-mediated antitumor activity

Abstract

Background: Major histocompatibility complex class I-related protein A and B (MICA/B) are ligands for the natural killer group 2 member D (NKG2D) receptor and are broadly expressed on tumor cells but minimally on normal tissues. When cytotoxic NKG2D-expressing immune cells engage MICA/B, the ligand-expressing cells are targeted for lysis. Cancer cells can evade NKG2D-mediated destruction by shedding MICA/B from their cell surface via proteases present in the tumor microenvironment. CLN-619 is a humanized IgG1 monoclonal antibody (mAb) which binds MICA/B and inhibits shedding resulting in accumulation of MICA/B on the tumor cell surface. CLN-619 may thereby have therapeutic effects in a broad range of malignancies by re-establishing the MICA/B-NKG2D axis to enable NKG2D-mediated, as well as Fc-gamma receptor-mediated, tumor cell lysis.

Methods: CLN-619 was characterized for binding epitope and affinity, effects on surface and soluble levels of MICA/B, and in vitro tumor cell killing. In mouse models, the mAb was tested for tumor growth inhibition. The contribution of the Fc-gamma (Fcγ) 1 domain to CLN-619 activity was also assessed.

Results: CLN-619 bound with high affinity to the alpha-3 domain of MICA/B without encumbering the interaction with NKG2D on natural killer cells. CLN-619 increased the level of cell surface expression of MICA/B and concomitantly decreased the levels of soluble MICA/B in cell culture assays. Treatment of cancer cell lines with CLN-619 induced antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis. CLN-619 resulted in potent inhibition of tumor growth in multiple xenograft models and increased survival of mice in a disseminated cancer model.

Conclusions: CLN-619 inhibited the shedding of MICA/B to effectively restore cytotoxic signaling pathways in immune cells. Potent antitumor activity of CLN-619 as a monotherapy was observed in several preclinical models. Activity of CLN-619 required a functional Fcγ1 domain, suggesting the requirement of simultaneous engagement of NKG2D and cluster of differentiation 16A (CD16A) on immune cells for optimal cytotoxicity. The preclinical data reported here support the assessment of CLN-619 in patients with cancer.

Keywords: Monoclonal antibody; Natural killer - NK.

Figure 1. Binding characteristics of CLN-619. CLN-619 binding to representative MICA allelic variants and the canonical MICB allelic variant as measured (A) by Octet (N=3; N refers to biological replicates throughout) and (B) by ELISA (N=2). CLN-619 had similar levels of binding to the various proteins; therefore, not all symbols are visible. (C) CLN-619 binding to 28 of the most common MICA allelic variants as measured by Luminex (N=2). 6D4 was used as a positive control. Error bars represent SEM. k_a, association constant; k_d, dissociation constant; K_D, equilibrium dissociation constant; OD, optical density; MFI, mean fluorescence intensity; MICA/B, major histocompatibility complex class I-related protein A/B; SEM, standard error of the mean.

Figure 2. CLN-619 co-crystal structure with MICA. X-ray crystal structures of the Fab fragment of CLN-619 in complex with the MICA*001 alpha-3 domain at 2.12 Å resolution. The orange box magnifies the binding interface of CLN-619, which is a discontinuous epitope comprising 19 amino acid residues exclusively in the alpha-3 domain of MICA. Å, angstrom; Fab, fragment antigen binding; MICA, major histocompatibility complex class I-related protein A.

Figure 3. CLN-619 modulates cell surface MICA/B through inhibition of shedding, while also engaging CD16A to enhance NKG2D signaling. (A) Cell surface levels of MICA/B were measured by flow cytometry. (B) Soluble MICA/B in the cell supernatant was measured by ELISA (N=3 or N=2). Geometric mean EC₅₀ values and 95% CIs are reported. (C) MICA-expressing target cells or control cells treated with serially titrated CLN-619 or CLN-619 DANA were co-cultured with NFAT-luciferase Jurkat cells expressing NKG2D or NKG2D and truncated CD16A with an E:T of 25:1 for 24 hours (N=3). T-test values from area under the curves are reported for each reporter line. Control CHO and isotype (500,000 pg/mL) treatment responses were close to baseline and cannot be visualized. E:T control background subtracted. Error bars represent SEM. EC₅₀, half-maximal effective concentration; CHO, Chinese hamster ovary; E:T, effector:target; IC₅₀, half-maximal inhibitory concentration; MICA/B, major histocompatibility complex class I-related protein A/B; NFAT, nuclear factor of activated T cells; NKG2D, natural killer group 2 member D; NS, not significant; RLU, relative luminesence units; sMICA/B, soluble MICA/B.

Figure 4. CLN-619 mediates NK cell activation and killing of MICA/B-expressing target cells. (A) An ELISA (R&D Systems) was used to measure IFN-γ in the supernatants collected from a 72 hour co-culture killing assay with NK cells and MICA/B expressing target cells (E:T 40:1) in the presence of serially titrated antibody in two donors (N=2). Representative curves are shown. (B) xCELLigence was used to measure MICA/B-expressing target cell death from experiment described in (A). EC₅₀ values were calculated for the last time point collected for each of the three runs. Representative curves are shown. For both panels, geometric mean EC₅₀ and 95% CI values are reported. Error bars represent SEM. EC₅₀, half-maximal effective concentration; E:T, effector:target; IFN, interferon; MICA/B, major histocompatibility complex class I-related protein A/B; NK, natural killer.

Figure 5. CLN-619 induces ADCC. MICA/B-expressing target cells treated with serially titrated antibody (CLN-619 or CLN-619 DANA) were co-cultured with NFAT-luciferase Jurkat cells expressing the human FcγRIIIa (CD16A) high affinity (V158) variant or low affinity (F158) variant at an E:T ratio of 10:1. Luminescence activity was quantified after 6 hours (N=3). Geometric mean EC₅₀ values and 95% CIs for CLN-619 are reported. E:T control background subtracted. Error bars represent SEM. ADCC, antibody-dependent cellular cytotoxicity; EC₅₀, half-maximal effective concentration; E:T, effector:target; F, phenylalanine; MICA/B, major histocompatibility complex class I-related protein A/B; NFAT, nuclear factor of activated T cells; RLU, relative luminescence units, V, valine.

Figure 6. CLN-619 promotes ADCP. Macrophage-mediated phagocytosis of fluorescently labeled target cells was measured by flow cytometry. Representative curves for treatment of HCC1534 and HCT-116 cells with CLN-619 in two donors (N=3). Cetuximab and isotype control antibody treatment were tested at the highest concentration. Geometric mean EC₅₀ values and 95% CIs are reported. Error bars represent SEM. E:T control background subtracted. ADCP, antibody-depedent cellular phagocytosis; EC₅₀, half-maximal effective concentration; E:T, effector:target; MFI, mean fluorescence intensity.

Figure 7. CLN-619 treatment results in potent antitumor effects in several preclinical mouse models. (A) BALB/c SCID mice were inoculated with PLC/PRF/5 cells (N=10/group). Mice were treated 3× weekly i.p. with CLN-619 at two dose levels. (B) BALB/c SCID mice were inoculated with HCC1534 cells (N=10/group). Mice were treated twice weekly i.p. with CLN-619 or CLN-619 DANA. (C) huIL15-NOG mice were engrafted with human PBMC and inoculated with A2058 cells (N=15/group). Mice were treated twice weekly i.p. with CLN-619 or CLN-619 DANA. *p=0.002, **p<0.0001. For all three efficacy studies, statistics represent two-way ANOVAs with multiple comparisons. (D) BALB/c SCID mice were inoculated with HCT-116 luciferase-tagged human colon cancer cells (N=10/group). Mice were treated twice weekly i.p. with CLN-619 or hIgG1. Error bars represent SEM. ANOVA, analysis of variance; i.p., intraperitoneal; PBMC, peripheral blood mononuclear cells.