Limits...
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

Expression and purification of the fusion proteins antiCD3Fv-⊿IL3 and the ds-antiCD3Fv-⊿IL3. Schematic of the expression plasmid for (A) antiCD3Fv-⊿IL3 and (B) ds-antiCD3Fv-⊿IL3, and structure of the fusion proteins for (C) antiCD3Fv-⊿IL3 and (D) ds-antiCD3Fv-⊿IL3. Note: the drawing is not to scale; asterisk (*) indicates the site of the disulfide bond. The fusion proteins were expressed in E. coli, purified by affinity chromatography, and analyzed by SDS-PAGE and immunoblotting. (E) Colloidal staining of a SDS-PAGE gel. Lane 1: antiCD3Fv-⊿IL3 in non-reducing loading buffer; lane 2: ds-antiCD3Fv-⊿IL3 in reducing loading buffer; lane 3: ds-antiCD3Fv-⊿IL3 in non-reducing loading buffer. (F) Immunoblot analysis of the SDS-PAGE gel with an anti-His-tag antibody. Lane 1: antiCD3Fv-⊿IL3 in non-reducing loading buffer; lane 2: ds-antiCD3Fv-⊿IL3 in reducing loading buffer; lane 3: ds-antiCD3Fv-⊿IL3 in non-reducing loading buffer.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Expression and purification of the fusion proteins antiCD3Fv-⊿IL3 and the ds-antiCD3Fv-⊿IL3. Schematic of the expression plasmid for (A) antiCD3Fv-⊿IL3 and (B) ds-antiCD3Fv-⊿IL3, and structure of the fusion proteins for (C) antiCD3Fv-⊿IL3 and (D) ds-antiCD3Fv-⊿IL3. Note: the drawing is not to scale; asterisk (*) indicates the site of the disulfide bond. The fusion proteins were expressed in E. coli, purified by affinity chromatography, and analyzed by SDS-PAGE and immunoblotting. (E) Colloidal staining of a SDS-PAGE gel. Lane 1: antiCD3Fv-⊿IL3 in non-reducing loading buffer; lane 2: ds-antiCD3Fv-⊿IL3 in reducing loading buffer; lane 3: ds-antiCD3Fv-⊿IL3 in non-reducing loading buffer. (F) Immunoblot analysis of the SDS-PAGE gel with an anti-His-tag antibody. Lane 1: antiCD3Fv-⊿IL3 in non-reducing loading buffer; lane 2: ds-antiCD3Fv-⊿IL3 in reducing loading buffer; lane 3: ds-antiCD3Fv-⊿IL3 in non-reducing loading buffer.

Mentions: The nucleotide sequences encoding the fusion proteins were verified by DNA sequencing. The last eight amino acids at the C-terminal of IL3 were entirely deleted. In addition, the Ser-100 of antiCD3VL and Gly-44 of antiCD3VH were successfully replaced by cysteines (Figure 1B). The fusion proteins were expressed by cosecretion of the two polypeptide chains (antiCD3VL-⊿IL3 or antiCD3*VL-⊿IL3 and antiCD3VH-⊿IL3-His or antiCD3*VH-⊿IL3-His) from Escherichia coli 16C9 cells as periplasmic native proteins (Figure 1A,B). Then, antiCD3VL-⊿IL3 and antiCD3VH-⊿IL3-His were folded to form fusion protein antiCD3Fv-⊿IL3 depending on the intermolecular force (Figure 1C) whereas the two cysteine-mutated polypeptide chains antiCD3*VL-⊿IL3 and antiCD3*VH-⊿IL3-His formed fusion protein ds-antiCD3Fv-⊿IL3 relying on the disulfide bonds in the periplasmic space (Figure 1D). The fusion proteins were released from the periplasmic space of E. coli by osmotic shock and purified by 6 × His-tag affinity chromatography. The yields of purified fusion proteins ranged from 1 to 2 mg/L of culture medium.Figure 1


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)

Expression and purification of the fusion proteins antiCD3Fv-⊿IL3 and the ds-antiCD3Fv-⊿IL3. Schematic of the expression plasmid for (A) antiCD3Fv-⊿IL3 and (B) ds-antiCD3Fv-⊿IL3, and structure of the fusion proteins for (C) antiCD3Fv-⊿IL3 and (D) ds-antiCD3Fv-⊿IL3. Note: the drawing is not to scale; asterisk (*) indicates the site of the disulfide bond. The fusion proteins were expressed in E. coli, purified by affinity chromatography, and analyzed by SDS-PAGE and immunoblotting. (E) Colloidal staining of a SDS-PAGE gel. Lane 1: antiCD3Fv-⊿IL3 in non-reducing loading buffer; lane 2: ds-antiCD3Fv-⊿IL3 in reducing loading buffer; lane 3: ds-antiCD3Fv-⊿IL3 in non-reducing loading buffer. (F) Immunoblot analysis of the SDS-PAGE gel with an anti-His-tag antibody. Lane 1: antiCD3Fv-⊿IL3 in non-reducing loading buffer; lane 2: ds-antiCD3Fv-⊿IL3 in reducing loading buffer; lane 3: ds-antiCD3Fv-⊿IL3 in non-reducing loading buffer.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Expression and purification of the fusion proteins antiCD3Fv-⊿IL3 and the ds-antiCD3Fv-⊿IL3. Schematic of the expression plasmid for (A) antiCD3Fv-⊿IL3 and (B) ds-antiCD3Fv-⊿IL3, and structure of the fusion proteins for (C) antiCD3Fv-⊿IL3 and (D) ds-antiCD3Fv-⊿IL3. Note: the drawing is not to scale; asterisk (*) indicates the site of the disulfide bond. The fusion proteins were expressed in E. coli, purified by affinity chromatography, and analyzed by SDS-PAGE and immunoblotting. (E) Colloidal staining of a SDS-PAGE gel. Lane 1: antiCD3Fv-⊿IL3 in non-reducing loading buffer; lane 2: ds-antiCD3Fv-⊿IL3 in reducing loading buffer; lane 3: ds-antiCD3Fv-⊿IL3 in non-reducing loading buffer. (F) Immunoblot analysis of the SDS-PAGE gel with an anti-His-tag antibody. Lane 1: antiCD3Fv-⊿IL3 in non-reducing loading buffer; lane 2: ds-antiCD3Fv-⊿IL3 in reducing loading buffer; lane 3: ds-antiCD3Fv-⊿IL3 in non-reducing loading buffer.
Mentions: The nucleotide sequences encoding the fusion proteins were verified by DNA sequencing. The last eight amino acids at the C-terminal of IL3 were entirely deleted. In addition, the Ser-100 of antiCD3VL and Gly-44 of antiCD3VH were successfully replaced by cysteines (Figure 1B). The fusion proteins were expressed by cosecretion of the two polypeptide chains (antiCD3VL-⊿IL3 or antiCD3*VL-⊿IL3 and antiCD3VH-⊿IL3-His or antiCD3*VH-⊿IL3-His) from Escherichia coli 16C9 cells as periplasmic native proteins (Figure 1A,B). Then, antiCD3VL-⊿IL3 and antiCD3VH-⊿IL3-His were folded to form fusion protein antiCD3Fv-⊿IL3 depending on the intermolecular force (Figure 1C) whereas the two cysteine-mutated polypeptide chains antiCD3*VL-⊿IL3 and antiCD3*VH-⊿IL3-His formed fusion protein ds-antiCD3Fv-⊿IL3 relying on the disulfide bonds in the periplasmic space (Figure 1D). The fusion proteins were released from the periplasmic space of E. coli by osmotic shock and purified by 6 × His-tag affinity chromatography. The yields of purified fusion proteins ranged from 1 to 2 mg/L of culture medium.Figure 1

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