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AntiCD3Fv fused to human interleukin-3 deletion variant redirected T cells against human acute myeloid leukemic stem cells.

Fan D, Li Z, Zhang X, Yang Y, Yuan X, Zhang X, Yang M, Zhang Y, Xiong D - J Hematol Oncol (2015)

Bottom Line: Thus, successful new therapeutic strategies for the treatment of AML should aim at eradicating LSCs.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.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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Experimental Hematology, Institute of Hematology & Hospital of Blood Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, 300020, People's Republic of China. fdm19691217@163.com.

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.

No MeSH data available.


Related in: MedlinePlus

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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Fig6: 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.

Mentions: We established a NOD/SCID mouse model bearing the human AML progenitor cell line KG1a to determine whether the fusion proteins also had an antitumor activity in vivo. The pathological finding showed KG1a tumor cells were in diffuse and intensive distribution, which indicated the subcutaneous KG1a xenotransplanted model was established successfully (Figure 6A). The expression level of CD123 on the surface of KG1a xenografts (70.620% ± 5.023%) was slightly reduced compared with KG1a cell line (77.875% ± 3.090%) (Figure 6B). Two doses (50 and 100 μg/mouse) of the fusion proteins were chosen to evaluate their ability to inhibit tumor growth in NOD/SCID mice. Animals were sacrificed when the tumors had grown to approximately 4,000 mm3 in size. As shown in Figure 6C, compared to the mice in the control group (untreated), the mice treated with different dosages of fusion proteins antiCD3Fv-⊿IL3 or ds-antiCD3Fv-⊿IL3 combined with pre-activated T lymphocytes showed a significant dose-related tumor regression. Furthermore, the ds fusion protein had a better antitumor activity than the parental fusion protein, when equivalent doses of ds fusion protein and the parent fusion protein were compared. The ability of tumor regression still exceeded its parental fusion protein slightly, even with half dose of the ds fusion protein (Figure 6C).Figure 6


AntiCD3Fv fused to human interleukin-3 deletion variant redirected T cells against human acute myeloid leukemic stem cells.

Fan D, Li Z, Zhang X, Yang Y, Yuan X, Zhang X, Yang M, Zhang Y, Xiong D - J Hematol Oncol (2015)

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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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4389834&req=5

Fig6: 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.
Mentions: We established a NOD/SCID mouse model bearing the human AML progenitor cell line KG1a to determine whether the fusion proteins also had an antitumor activity in vivo. The pathological finding showed KG1a tumor cells were in diffuse and intensive distribution, which indicated the subcutaneous KG1a xenotransplanted model was established successfully (Figure 6A). The expression level of CD123 on the surface of KG1a xenografts (70.620% ± 5.023%) was slightly reduced compared with KG1a cell line (77.875% ± 3.090%) (Figure 6B). Two doses (50 and 100 μg/mouse) of the fusion proteins were chosen to evaluate their ability to inhibit tumor growth in NOD/SCID mice. Animals were sacrificed when the tumors had grown to approximately 4,000 mm3 in size. As shown in Figure 6C, compared to the mice in the control group (untreated), the mice treated with different dosages of fusion proteins antiCD3Fv-⊿IL3 or ds-antiCD3Fv-⊿IL3 combined with pre-activated T lymphocytes showed a significant dose-related tumor regression. Furthermore, the ds fusion protein had a better antitumor activity than the parental fusion protein, when equivalent doses of ds fusion protein and the parent fusion protein were compared. The ability of tumor regression still exceeded its parental fusion protein slightly, even with half dose of the ds fusion protein (Figure 6C).Figure 6

Bottom Line: Thus, successful new therapeutic strategies for the treatment of AML should aim at eradicating LSCs.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.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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Experimental Hematology, Institute of Hematology & Hospital of Blood Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, 300020, People's Republic of China. fdm19691217@163.com.

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.

No MeSH data available.


Related in: MedlinePlus