Targeting human C-type lectin-like molecule-1 (CLL1) with a bispecific antibody for immunotherapy of acute myeloid leukemia

Abstract

Acute myeloid leukemia (AML), which is the most common acute adult leukemia and the second most common pediatric leukemia, still has a poor prognosis. Human C-type lectin-like molecule-1 (CLL1) is a recently identified myeloid lineage restricted cell surface marker, which is overexpressed in over 90% of AML patient myeloid blasts and in leukemic stem cells. Here, we describe the synthesis of a novel bispecific antibody, αCLL1-αCD3, using the genetically encoded unnatural amino acid, p-acetylphenylalanine. The resulting αCLL1-αCD3 recruits cytotoxic T cells to CLL1 positive cells, and demonstrates potent and selective cytotoxicity against several human AML cell lines and primary AML patient derived cells in vitro. Moreover, αCLL1-αCD3 treatment completely eliminates established tumors in an U937 AML cell line xenograft model. These results validate the clinical potential of CLL1 as an AML-specific antigen for the generation of a novel immunotherapeutic for AML.

Keywords: CLL1; acute myeloid leukemia; bispecific antibodies; cancer immunotherapy; unnatural amino acids.

Figure 1

Synthesis and characterization of αCLL1-αCD3 and αCD33-αCD3. Synthetic scheme for the BiFabs (A); SDS-PAGE gel of BiFabs: lane 1 protein ladder; lanes 2–3 αCD33-αCD3 and αCLL1-αCD3 under non-reducing condition, respectively; lanes 4–5 αCD33-αCD3 and αCLL1-αCD3 under reducing condition, respectively (B); Binding of Fab mutants and BiFabs to human AML cell line U937(C) and to human T cells (D); goat anti-human Kappa-RPE (Southern Biotech Associate) was used as the secondary antibody for cell labeling.

Figure 2

In vitro cytotoxicity of αCLL1-αCD3 and αCD33-αCD3 redirecting healthy PBMCs against various human AML cell lines - U937 (A) and HL60 (B) after 24h or 48h incubations. Cytotoxicity curves of αCD33-αCD3 against U937, Kasumi-3 and KG-1A (C), and of αCLL1-αCD3 against U937, Kasumi-3 and KG-1A (D) after a 24h incubation period. In all experiments, target cells were stained by either PKH26 or CellVue Claret Far Red (Sigma-Aldrich) and incubated with PBMCs at a 1/10 ratio. Each data point represents a mean of triplicate samples. Error bars are representative of standard deviation.

Figure 3

Ex vivo cytotoxicity of αCLL1-αCD3 and αCD33-αCD3 against primary AML patient samples. A) Relative viability of AML1 (CD33⁻/CLL1⁺) blasts treated with αCD33-αCD3, αCLL1-αCD3, non-conjugated Fab mixture of αCD33 + αCD3, or non-conjugated Fab mixture of αCLL1+ αCD3 for 24hr; B) time-dependent ex vivo viability of AML5 (CD33⁺/CLL1⁺) blasts treated with 1 nM αCD33-αCD3 or αCLL1-αCD3; relative viability of (C) AML3 (CD33⁺/CLL1⁺) and (D) AML7 (CD33⁺/CLL1⁺) blasts treated with αCD33-αCD3 or αCLL1-αCD3 with non-activated autologous T cells in PBMCs for 96hr (filled) or with ex vivo expanded autologous T cells for 48hr (open).

Figure 4

In vivo anti-tumor activity of αCLL1-αCD3 and αCD33-αCD3. (A) U937 cells and PBMCs were injected in NSG mice on Day 0. Upon observation of a palpable tumor, ex vivo expanded T cells were injected, and 24h later, animals were dosed daily with αCLL1-αCD3 (1mg/kg), αCD33-αCD3 (1mg/kg), or PBS control for 10 days. (B) T cell infiltrates in U937 tumors treated with αCLL1-αCD3 (1mg/kg), αCD33-αCD3 (1mg/kg), or PBS.