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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

Comparison of the stability of fusion proteins antiCD3Fv-⊿IL3 and ds-antiCD3Fv-⊿IL3in 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 t0 (initial time), which was set at 100%. The data shown represent the averages of three independent experiments.
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Fig3: Comparison of the stability of fusion proteins antiCD3Fv-⊿IL3 and ds-antiCD3Fv-⊿IL3in 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 t0 (initial time), which was set at 100%. The data shown represent the averages of three independent experiments.

Mentions: The stability of the fusion proteins in vitro was determined by analysis of their binding to target cells after incubation in PBS containing 0.2% (w/v) human serum albumin (HSA) at 37°C for several hours. The results showed that the binding capacity of the fusion proteins antiCD3Fv-⊿IL3 was reduced by nearly 50% after incubation for 6 h and 80% for 72 h, while the binding capacity of the ds-antiCD3Fv-⊿IL3 remained more than 90% after 72 h (Figure 3A,B).Figure 3


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)

Comparison of the stability of fusion proteins antiCD3Fv-⊿IL3 and ds-antiCD3Fv-⊿IL3in 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 t0 (initial time), which was set at 100%. The data shown represent the averages of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Comparison of the stability of fusion proteins antiCD3Fv-⊿IL3 and ds-antiCD3Fv-⊿IL3in 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 t0 (initial time), which was set at 100%. The data shown represent the averages of three independent experiments.
Mentions: The stability of the fusion proteins in vitro was determined by analysis of their binding to target cells after incubation in PBS containing 0.2% (w/v) human serum albumin (HSA) at 37°C for several hours. The results showed that the binding capacity of the fusion proteins antiCD3Fv-⊿IL3 was reduced by nearly 50% after incubation for 6 h and 80% for 72 h, while the binding capacity of the ds-antiCD3Fv-⊿IL3 remained more than 90% after 72 h (Figure 3A,B).Figure 3

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