title: AntiCD3Fv fused to human interleukin-3 deletion variant redirected T cells against human acute myeloid leukemic stem cells.
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.
authors: 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.
authors: Dongmei
authors: D
authors: Fan
authors: 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. lizhenvvv@163.com.
authors: Zhenzhen
authors: Z
authors: Li
authors: 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. zhxlongmu@126.com.
authors: Xiaolong
authors: X
authors: Zhang
authors: School of Pharmacy, Tianjin Medical University, Tianjin, 300070, People's Republic of China. yangyvjian@gmail.com.
authors: Yuqi
authors: Y
authors: Yang
authors: 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. xfyuanca@sohu.com.
authors: Xiangfei
authors: X
authors: Yuan
authors: 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. zxlics@yeah.net.
authors: Xiuli
authors: X
authors: Zhang
authors: 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. my1970@sina.com.
authors: Ming
authors: M
authors: Yang
authors: Central Hospital of Karamay, Karamay, Xinjiang, 834000, China. yz5168813@sohu.com.
authors: Yizhi
authors: Y
authors: Zhang
authors: 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. dsxiong@ihcams.ac.cn.
authors: Dongsheng
authors: D
authors: Xiong
mesh_terms: D000818:Animals; D017252:CD3 Complex / Q000276:immunology*; D045744:Cell Line, Tumor; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D007167:Immunotherapy / Q000379:methods*; D053650:Interleukin-3 Receptor alpha Subunit / Q000276:immunology*; D015470:Leukemia, Myeloid, Acute / Q000276:immunology* / Q000473:pathology; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D014411:Neoplastic Stem Cells / Q000276:immunology* / Q000473:pathology; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000494:pharmacology*; D013601:T-Lymphocytes / Q000276:immunology; D023041:Xenograft Model Antitumor Assays
chemical_list: D017252:CD3 Complex; C115022:IL3RA protein, human; D053650:Interleukin-3 Receptor alpha Subunit; D011993:Recombinant Fusion Proteins