A novel IgG-based FLT3xCD3 bispecific antibody for the treatment of AML and B-ALL

Abstract

Background: In lymphoid malignancies, the introduction of chimeric antigen receptor T (CAR-T) cells and bispecific antibodies (bsAbs) has achieved remarkable clinical success. However, such immunotherapeutic strategies are not yet established for acute myeloid leukemia (AML), the most common form of acute leukemia in adults. Common targets in AML such as CD33, CD123, and CLEC12A are highly expressed on both AML blasts and on normal myeloid cells and hematopoietic stem cells (HSCs), thereby raising toxicity concerns. In B-cell acute lymphoblastic leukemia (B-ALL), bsAbs and CAR-T therapy targeting CD19 and CD22 have demonstrated clinical success, but resistance via antigen loss is common, motivating the development of agents focused on alternative targets. An attractive emerging target is FLT3, a proto-oncogene expressed in both AML and B-ALL, with low and limited expression on myeloid dendritic cells and HSCs.

Methods: We developed and characterized CLN-049, a T cell-activating bsAb targeting CD3 and FLT3, constructed as an IgG heavy chain/scFv fusion. CLN-049 binds the membrane proximal extracellular domain of the FLT3 protein tyrosine kinase, which facilitates the targeting of leukemic blasts regardless of FLT3 mutational status. CLN-049 was evaluated for preclinical safety and efficacy in vitro and in vivo.

Results: CLN-049 induced target-restricted activation of CD4+ and CD8+ T cells. AML cell lines expressing a broad range of surface levels of FLT3 were efficiently lysed on treatment with subnanomolar concentrations of CLN-049, whereas FLT3-expressing hematopoietic progenitor cells and dendritic cells were not sensitive to CLN-049 killing. Treatment with CLN-049 also induced lysis of AML and B-ALL patient blasts by autologous T cells at the low effector-to-target ratios typically observed in patients with overt disease. Lysis of leukemic cells was not affected by supraphysiological levels of soluble FLT3 or FLT3 ligand. In mouse xenograft models, CLN-049 was highly active against human leukemic cell lines and patient-derived AML and B-ALL blasts.

Conclusions: CLN-049 has a favorable efficacy and safety profile in preclinical models, warranting evaluation of its antileukemic activity in the clinic.

Keywords: T-Lymphocytes; immunity; immunotherapy.

Figure 1

Structure and characterization of CLN-049 (A) Design of CLN-049. (B) SDS-PAGE analysis of CLN-049 under reducing and non-reducing conditions. (C) SEC trace of CLN-049 following Protein A-based affinity purification. (D) Mass spectrum of CLN-049. (E) Heat flux curve from DSC evaluation of CLN-049 determining three melting temperatures for CLN-049. DSC, differentiating scanning calorimetry; SDS-PAGE, sodium dodecyl-sulfate polyacrylamide gel electrophoresis; SEC, size exclusion chromatography.

Figure 2

Binding characteristics of CLN-049. (A) Summary of Biacore binding data of CLN-049 using FLT3 and FLT3-related proteins. (B) Binding of CLN-049 to CHO cells expressing WT FLT3 or FLT3 N399D. (C) Human FLT3 ligand-receptor complex in dimeric form, based on PDB 3QS9. Dimeric ligands are colored in raspberry. Receptor ectodomains are colored as follows: domain one in yellow, domain two in green, domain three in cyan, domain four in blue, and domain five in gray. N399 in domain four is colored in red. (D) Binding of CLN-049 to FLT3+ NALM-16 cells and CD3+Jurkat cells by flow cytometry. (E) Flow cytometry plots depicting HLA-DR+ cells from PBMC of a representative donor stained with PE-labeled FLT3 mAb clone BV10 (Untreated), an APC-labeled IgG₁ isotype control antibody, or APC-labeled CLN-049. The panel below depicts quantification across three donors, with the percentage of CLN-049 calculated as %CLN-049+ - %IC+. (F) Histograms showing purified T cells from a representative donor stained with APC-labeled IgG₁ isotype control antibody (red) or APC-labeled CLN-049 (blue). The panel below shows quantification across three donors, where the percentage of CLN-049 is calculated as %CLN-049+ - %IC+. (G) Sandwich ELISA with FLT3 capture, incubation with titrations of CLN-049, and CD3-biotin based detection. APC, allophycocyanin; DC, dendritic cell; PBMC, peripheral blood mononuclear cells.

Figure 3

CLN-049 potently induces T cell activation and TDCC. (A) FLT3 receptor number on 14 AML cell lines and 20 patient BM blast samples. (B) AML cell lines were labeled with cell proliferation dye, cocultured with PBMC from three different healthy donors at an E:T ratio of 2:1, in the presence or absence of the indicated concentrations of CLN-049 for 72 hours. Shown are lysis curves, as flow cytometrically assessed by 7-AAD uptake in AML cells and CD69 expression profiles as marker for activation of CD4+ and CD8+ T cells. (C) Plot of FLT3 receptor number relative to CLN-049 mediated T cell activation killing and target cell lysis as measured by flow cytometry. Numbers identify the different AML cell lines used as targets. Correlation constants were calculated using log-normalized values for both FLT3 receptor number and EC₅₀ values. (D) MOLM-13 and Jurkat cell lysis was determined as described in (B). (E) Supernatants from cocultures of MOLM-13 cells or U-937 cells with PBMC as in (B) were analyzed for the indicated cytokines by Luminex. AML, acute myeloid leukemia; BM, bone marrow; PBMC, peripheral blood mononuclear cells; TDCC, T cell dependent cellular cytotoxicity; 7-AAD, 7-aminoactinomycin D.

Figure 4

In vitro safety of CLN-049 (A) FLT3 receptor number of normal cells shown in red, compared with FLT3 receptor number of AML cell lines in black. Numbers show the frequency of live events of the indicated cell type. Each point represents data from an individual donor. (B) PBMC from healthy donors were incubated with increasing concentrations of CLN-049 for 72 hours followed by assessment of pDCs lysis by flow cytometry. (C) BM cells from healthy donors were incubated with increasing concentrations of CLN-049 for 72 hours followed by assessment of CD34+ cells lysis by flow cytometry. (D) Purified T cells from the indicated donors were incubated with increasing concentrations of the indicated CD3-specific antibodies for 72 hours followed by assessment of CD4+ and CD8+ T cell activation via CD69 analysis by flow cytometry. (E) PBMC from healthy donors were incubated with increasing concentrations of the indicated CD3-specific antibodies for 72 hours in the presence or absence of MOLM-13 cells. Subsequently, CD4+ and CD8+ T cell activation was assessed via analysis of CD69 expression by flow cytometry. AML, acute myeloid leukemia; BM, bone marrow; pDCs, plasmacytoid dendritic cells; PBMC, peripheral blood mononuclear cells.

Figure 5

In vivo activity of CLN-049 (A, B) Study designs of CLN-049 administered to NCG mice engrafted with 1×10⁵ MOLM-13 AML cells and 2×10⁷ human PBMC, consisting of (A) a PK cohort and (B) an efficacy cohort. (C) PK curve of CLN-049 injected intravenously at 0.01 mg/kg. (D) MOLM-13 counts in peripheral blood at day 15. (E) Kaplan-Meier survival curves. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test (D) or Mantel-Cox test (E). AML, acute myeloid leukemia; ANOVA, analysis of variance; PBMC, peripheral blood mononuclear cells; PK, pharmacokinetics.

Figure 6

Cytotoxicity against primary AML cells (A) PBMC from three AML patients were incubated with increasing doses of CLN-049 for 72 hours followed by assessment of target cell lysis by flow cytometry. (B) NSG mice were coengrafted with 8×10⁶ PBMC from an AML patient and 8×10⁶ PBMC from a healthy donor, and then treated with CLN-049 or a control antibody (MOPCxCD3) on days 0, 3, and 10 (1 mg/kg on day 0, and 0.5 mg/kg on days 3 and 10). Leukemic burden was evaluated by flow cytometry of BM on day 24 and quantified by the ratio of human AML cells (hCD45+hCD33+) to murine hematopoietic cells (mCD45+). ****p<0.0001. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test (B). ANOVA, analysis of variance; AML, acute myeloid leukemia; BM, bone marrow.

Figure 7

Efficacy of CLN-049 in B-ALL (A) Fifteen samples of B-ALL patients were evaluated for CD20, CD22, and FLT3 expression by flow cytometry. B-ALL blasts were identified as CD19+. Shown in each graph is a rank-ordered distribution of expression patterns for each target. (B) Representative donor with low expression of CD20 and CD22 and high expression of FLT3. (C) PBMC from two B-ALL patients were incubated with increasing doses of CLN-049 for 72 hours followed by assessment of target cell lysis by flow cytometry. (D) NSG mice were coengrafted with 4×10⁶ PBMC from a B-ALL patient and 10×10⁶ PBMC from a healthy donor, then treated with CLN-049 or a control antibody (MOPCxCD3) on days 7 and 11 (1.5 mg/kg). Leukemic burden was evaluated by flow cytometry of the BM on day 17 by the ratio of human B-ALL cells (hCD45+hCD33+) to murine hematopoietic cells (mCD45+). **p<0.01. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test (D). ANOVA, analysis of variance; B-ALL, B-cell acute lymphoblastic leukemia; BM, bone marrow.