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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

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.
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Fig2: 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.

Mentions: Both antiCD3Fv-⊿IL3 and ds-antiCD3Fv-⊿IL3 bind to CD123-positive KG1a cells and CD3-positive Jurkat cells with similar efficiency as their parental monoclonal antibodies CD123 and HIT3a to the respective target cells (Figure 2A,B,E,F). Furthermore, both fusion proteins were able to partly reduce the binding efficiency of the parental monoclonal antibodies HIT3a and CD123 McAb to Jurkat and KG1a, respectively, in a competitive binding assay (Figure 2C,D,G,H). No significant decrease in binding activity of ds-antiCD3Fv-⊿IL3 fusion protein was detected compared to the parental fusion protein antiCD3Fv-⊿IL3.Figure 2


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)

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

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Fig2: 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.
Mentions: Both antiCD3Fv-⊿IL3 and ds-antiCD3Fv-⊿IL3 bind to CD123-positive KG1a cells and CD3-positive Jurkat cells with similar efficiency as their parental monoclonal antibodies CD123 and HIT3a to the respective target cells (Figure 2A,B,E,F). Furthermore, both fusion proteins were able to partly reduce the binding efficiency of the parental monoclonal antibodies HIT3a and CD123 McAb to Jurkat and KG1a, respectively, in a competitive binding assay (Figure 2C,D,G,H). No significant decrease in binding activity of ds-antiCD3Fv-⊿IL3 fusion protein was detected compared to the parental fusion protein antiCD3Fv-⊿IL3.Figure 2

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