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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

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.
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Fig4: 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.

Mentions: A non-radioactive cytotoxicity assay was performed to determine the ability of the fusion proteins antiCD3Fv-⊿IL3 and ds-antiCD3Fv-⊿IL3 to induce lysis of CD123+ tumor cells in the presence of pre-activated human T cells. The fusion proteins antiCD3Fv-⊿IL3 and ds-antiCD3Fv-⊿IL3 appeared to be potent in retargeting T cell lysis of the CD123-positive KG1a cells (Figure 4A,B,C), whereas CD123-negative cells THP1 and NB4 were not lysed under the same conditions (Figure 4D,E). Correspondingly, the ratio of apoptotic cells in the group of antiCD3Fv-⊿IL3 (65.633% ± 3.807%) and ds-antiCD3Fv-⊿IL3 (67.733% ± 3.821%) were increased when combined with pre-activated human T cells (Figure 4F,G). Additional movie files show this process of target cell lysis in more detail (see Additional file 1: Movie 1, Additional file 2: Movie 2, Additional file 3: Figure S1, and Additional file 4: Figure S2). Lysis of target cells mediated by the fusion protein or ds fusion protein increased in a dose-dependent manner: increasing either the concentration of fusion proteins or the ratio of effector to target cells resulted in enhanced target cell cytotoxicity (Figure 4A,B). The cytotoxic activity was not enhanced when an equal amount of the antiCD3/antiCD19 diabody was added, because CD19 was not expressed on the KG1a cells (Figure 4C). There was no statistical difference between the parental fusion protein and the ds fusion protein (500 ng/mL) in mediating the lysis of KG1a cells at various E:T ratios (Figure 4C).Figure 4


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)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4389834&req=5

Fig4: 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.
Mentions: A non-radioactive cytotoxicity assay was performed to determine the ability of the fusion proteins antiCD3Fv-⊿IL3 and ds-antiCD3Fv-⊿IL3 to induce lysis of CD123+ tumor cells in the presence of pre-activated human T cells. The fusion proteins antiCD3Fv-⊿IL3 and ds-antiCD3Fv-⊿IL3 appeared to be potent in retargeting T cell lysis of the CD123-positive KG1a cells (Figure 4A,B,C), whereas CD123-negative cells THP1 and NB4 were not lysed under the same conditions (Figure 4D,E). Correspondingly, the ratio of apoptotic cells in the group of antiCD3Fv-⊿IL3 (65.633% ± 3.807%) and ds-antiCD3Fv-⊿IL3 (67.733% ± 3.821%) were increased when combined with pre-activated human T cells (Figure 4F,G). Additional movie files show this process of target cell lysis in more detail (see Additional file 1: Movie 1, Additional file 2: Movie 2, Additional file 3: Figure S1, and Additional file 4: Figure S2). Lysis of target cells mediated by the fusion protein or ds fusion protein increased in a dose-dependent manner: increasing either the concentration of fusion proteins or the ratio of effector to target cells resulted in enhanced target cell cytotoxicity (Figure 4A,B). The cytotoxic activity was not enhanced when an equal amount of the antiCD3/antiCD19 diabody was added, because CD19 was not expressed on the KG1a cells (Figure 4C). There was no statistical difference between the parental fusion protein and the ds fusion protein (500 ng/mL) in mediating the lysis of KG1a cells at various E:T ratios (Figure 4C).Figure 4

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