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. 2015 Feb 28;8:18.
doi: 10.1186/s13045-015-0109-5.

AntiCD3Fv Fused to Human interleukin-3 Deletion Variant Redirected T Cells Against Human Acute Myeloid Leukemic Stem Cells

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Free PMC article

AntiCD3Fv Fused to Human interleukin-3 Deletion Variant Redirected T Cells Against Human Acute Myeloid Leukemic Stem Cells

Dongmei Fan et al. J Hematol Oncol. .
Free PMC article

Abstract

Background: Leukemic stem cells (LSCs) are frequently seen as a cause of treatment failure and relapse in patients with acute myeloid leukemia (AML). Thus, successful new therapeutic strategies for the treatment of AML should aim at eradicating LSCs. The identification of targets on the cell surface of LSCs is getting more and more attention. Among these, CD123, also known as the interleukin-3 (IL3)-receptor α chain, has been identified as a potential immunotherapeutic target due to its overexpression on LSCs in AML as well as on AML blasts, rather than normal hematopoietic stem cells.

Methods: We constructed a CD123-targeted fusion protein antiCD3Fv-⊿IL3, with one binding site for T cell antigen receptor (TCRCD3) and the other for CD123, by recombinant gene-engineering technology. Cysteine residues were introduced into the V domains of the antiCD3Fv segment to enhance its stability by locking the two chains of Fv together with disulfide covalent bonds. The stability and cytotoxicity of the two fusion proteins were detected in vitro and in vivo.

Results: Both fusion proteins were produced and purified from Escherichia coli 16C9 cells with excellent yields in fully active forms. High-binding capability was observed between these two fusion proteins and human IL3R, leading to the specific lysis of CD123-expressing cell lines KG1a; also, mononuclear cells from primary AML patients were inhibited in a colony forming assay in vitro, presumably by redirecting T lymphocytes in vitro. In addition, they displayed an antileukemic activity against KG1a xenografts in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice, especially disulfide-stabilized (ds)-antiCD3Fv-⊿IL3 for its improved stability.

Conclusions: These results suggest that both fusion proteins display the antileukemic activity against CD123-expressing cell lines as well as leukemic progenitors in vitro and in vivo, especially ds-antiCD3Fv-⊿IL3. They could be the promising candidates for future immunotherapy of AML.

Figures

Figure 1
Figure 1
Expression and purification of the fusion proteins antiCD3Fv-IL3 and the ds-antiCD3Fv-IL3. Schematic of the expression plasmid for (A) antiCD3Fv-⊿IL3 and (B) ds-antiCD3Fv-⊿IL3, and structure of the fusion proteins for (C) antiCD3Fv-⊿IL3 and (D) ds-antiCD3Fv-⊿IL3. Note: the drawing is not to scale; asterisk (*) indicates the site of the disulfide bond. The fusion proteins were expressed in E. coli, purified by affinity chromatography, and analyzed by SDS-PAGE and immunoblotting. (E) Colloidal staining of a SDS-PAGE gel. Lane 1: antiCD3Fv-⊿IL3 in non-reducing loading buffer; lane 2: ds-antiCD3Fv-⊿IL3 in reducing loading buffer; lane 3: ds-antiCD3Fv-⊿IL3 in non-reducing loading buffer. (F) Immunoblot analysis of the SDS-PAGE gel with an anti-His-tag antibody. Lane 1: antiCD3Fv-⊿IL3 in non-reducing loading buffer; lane 2: ds-antiCD3Fv-⊿IL3 in reducing loading buffer; lane 3: ds-antiCD3Fv-⊿IL3 in non-reducing loading buffer.
Figure 2
Figure 2
Analysis of specific binding of fusion proteins antiCD3Fv-IL3 and ds-antiCD3Fv-IL3 to Jurkat and KG1a, as well as the competitive binding activity with monoclonal antibody HIT3a or CD123. (A, B) Jurkat cells were incubated with fusion proteins (A) antiCD3Fv-⊿IL3 or (B) ds-antiCD3Fv-⊿IL3. (E-F) KG1a cells were incubated with (E) antiCD3Fv-⊿IL3 or (F) ds-antiCD3Fv-⊿IL3. Note: the gate represented the positive rate of binding of fusion proteins. In the competitive binding assay, (C, D) Jurkat cells were first incubated with ((C): c) antiCD3Fv-⊿IL3 or ((D): c) ds-antiCD3Fv-⊿IL3 and HIT3a. (G, H) KG1a cells were first incubated with ((G): c) antiCD3Fv-⊿IL3 or ((H): c) ds-antiCD3Fv-⊿IL3 and mAb CD123, then incubated with an FITC-conjugated antimouse IgG. Control groups only reacted with (a.) PBS and (b.) parent mAb IgG. Note: the gate represented the binding rate of the line of c with monoclonal antibody HIT3a or CD123.
Figure 3
Figure 3
Comparison of the stability of fusion proteins antiCD3Fv-IL3 and ds-antiCD3Fv-IL3 in vitro . The stability was determined by testing binding activity of fusion proteins to (A) CD3-positive Jurkat cells or (B) CD123-positive KG1a cells after incubation in 0.2% HSA at 37°C for a prolonged time period. Data were normalized to t 0 (initial time), which was set at 100%. The data shown represent the averages of three independent experiments.
Figure 4
Figure 4
Cytotoxicity of IL2 pre-activated human T cells to KG1a cells in different effector to target (E/T) ratios mediated by different concentrations of fusion proteins in a non-radioactive cytotoxicity assay. (A) Cytotoxicity of T cells in the presence of antiCD3Fv-⊿IL3. (B) Cytotoxicity of T cells in the presence of ds-antiCD3Fv-⊿IL3. Concentrations of fusion proteins were different (500, 50, 5 ng/mL). E/T cell ratios ranged from 25:1 to 3:1. (C) Lysis of target cells by T cells mediated by PBS, ds-antiCD3Fv-⊿IL3, antiCD3Fv-⊿IL3, and the control diabody antiCD19 × antiCD3. The concentration of all fusion proteins was 500 ng/mL. CD123-negative cell line (D) THP1 and (E) NB4 were included as a control. (F, G) Ratios of apoptotic cells mediated by fusion proteins (500 ng/mL) combined with pre-activated T cells at E/T ratio of 25:1. PBS and antiCD19 × antiCD3 are used as a control. **P < 0.01 vs PBS. Data shown are the mean ± SD of three repeated experiments.
Figure 5
Figure 5
Cytotoxicity of IL2 pre-activated human T cells mediated by the fusion proteins against AML leukemic progenitors in a methylcellulose colony-forming assay. (A) AML cells from six patients were incubated in serum-free IMDM for 24 h in the presence or absence of different fusion proteins (500 ng/mL) combined with pre-activated T cells at E/T ratio of 25:1, then plated in AML-CFC assays to evaluate the relative cytotoxicity of T cells mediated by the fusion proteins against these leukemic progenitors. (B-F) showed typical colonies of different groups. (B) PBS, (C) antiCD3Fv-⊿IL3, or (D) ds-antiCD3Fv-⊿IL3 alone was used as control group. (E) Cytotoxicity of T cells against leukemic progenitors in the presence of antiCD3Fv-⊿IL3. (F) Cytotoxicity of T cells against leukemic progenitors in the presence of ds-antiCD3Fv-⊿IL3.
Figure 6
Figure 6
Antitumor effect of fusion proteins antiCD3Fv-IL3 and ds-antiCD3Fv-IL3 in KG1a xenografted NOD/SCID mice. (A) H&E staining showed the KG1a tumor cells were diffused and in intensive distribution, indicating subcutaneous KG1a xenotransplanted model was established successfully. (B) The expression level of CD123 on the surface of KG1a xenografts and KG1a cell line were analyzed by FACS. (C) A mixture of T cells and fusion proteins antiCD3Fv-⊿IL3 or ds-antiCD3Fv-⊿IL3 in different concentrations (50 and 100 μg/mouse) were injected intravenously 6 days later, when the solid tumors reached 80–100 mm3 in size, once per week for 2 weeks. PBS, antiCD3Fv-⊿IL3, ds-antiCD3Fv-⊿IL3, and T cells alone, and T cells combined with antiCD19 × antiCD3 diabody, were used as control groups. The size of the xenografts was measured every 3 days. Error bars indicate SD (n = 5). **P < 0.01 vs PBS.

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