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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 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.
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Fig5: 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.

Mentions: The peripheral blood mononuclear cells from six newly diagnosed AML patients were incubated in serum-free IMDM for 24 h in the presence or absence of different fusion proteins combined with pre-activated T cells. Treated and untreated cells were then plated in AML-colony-forming cell (CFC) assays to evaluate the relative cytotoxicity of T cells mediated by the fusion proteins against these leukemic progenitors. AML-CFCs were detected in control (including PBS-treated or fusion proteins without pre-activated T cells) cultures of all six AML samples, ranging from 112 to 490 AML-CFCs/104 cells (Figure 5A,B,C,D). After 24-h exposure to fusion proteins with pre-activated T cells, the mean percentages of kill of AML-CFCs were 53.3% and 52.8%, respectively, for antiCD3Fv-⊿IL3 and ds-antiCD3Fv-⊿IL3, (range, 33.9% ~ 63.4%) (Figure 5A,E,F). There was no statistical difference between the parental fusion protein and the ds fusion protein (500 ng/mL) in mediating the inhibition of colony formation of AML leukemic progenitors in vitro (Figure 5A). These findings indicate that the two fusion proteins can preferentially retarget T cells to AML progenitor cells.Figure 5


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

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4389834&req=5

Fig5: 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.
Mentions: The peripheral blood mononuclear cells from six newly diagnosed AML patients were incubated in serum-free IMDM for 24 h in the presence or absence of different fusion proteins combined with pre-activated T cells. Treated and untreated cells were then plated in AML-colony-forming cell (CFC) assays to evaluate the relative cytotoxicity of T cells mediated by the fusion proteins against these leukemic progenitors. AML-CFCs were detected in control (including PBS-treated or fusion proteins without pre-activated T cells) cultures of all six AML samples, ranging from 112 to 490 AML-CFCs/104 cells (Figure 5A,B,C,D). After 24-h exposure to fusion proteins with pre-activated T cells, the mean percentages of kill of AML-CFCs were 53.3% and 52.8%, respectively, for antiCD3Fv-⊿IL3 and ds-antiCD3Fv-⊿IL3, (range, 33.9% ~ 63.4%) (Figure 5A,E,F). There was no statistical difference between the parental fusion protein and the ds fusion protein (500 ng/mL) in mediating the inhibition of colony formation of AML leukemic progenitors in vitro (Figure 5A). These findings indicate that the two fusion proteins can preferentially retarget T cells to AML progenitor cells.Figure 5

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