A Unique Human Immunoglobulin Heavy Chain Variable Domain-Only CD33 CAR for the Treatment of Acute Myeloid Leukemia

Frontiers in Oncology

Dimiter S. Dimitrov¹  Tianlei Ying²  Darong Wu³  Boro Dropulic³  Dina Schneider³  Ying Xiong³  Peirong Hu³  Zhongyu Zhu⁴  Weizao Chen⁴  Rimas J. Orentas⁵ 

[1] Center for Antibody Therapeutics, Department of Medicine, School of Medicine, University of Pittsburgh, Pittsburgh, PA, United States;Key Laboratory of Medical Molecular Virology, Ministries of Education and Health, School of Basic Medical Sciences, Fudan University, Shanghai, China;Lentigen, A Miltenyi Biotec Company, Gaithersburg, MD, United States;Protein Interactions Section, Cancer and Inflammation Program, National Cancer Institute (NCI), National Institutes of Health (NIH), Frederick, MD, United States;Seattle Children's Research Institute, Seattle, WA, United States;

关键词: AML;    lentiviral vector;    CAR-T;    CD33;    VH;    immunotherapy;   

DOI  :  10.3389/fonc.2018.00539

来源: DOAJ

【 摘 要 】

Acute myeloid leukemia (AML) remains a challenging pediatric and adult disease. Given the elevated expression of the CD33 antigen on leukemic blasts, therapeutic approaches to AML now feature the approved antibody drug conjugate (Mylotarg, GO) and investigational CART cell approaches incorporating CD33-binding domains derived from humanized scFvs. We designed a functional chimeric antigen receptor utilizing a human targeting sequence, derived from a heavy chain variable domain, termed CAR33VH. Lentiviral-based expression vectors which encoded CAR constructs incorporating the novel binding domain (CAR33VH), or the My96 scFv control binder (My96CAR) in frame with a CD8 hinge and transmembrane domain, a 4-1BB costimulatory domain and a CD3 zeta activation domain, were transduced into primary human CD4+ and CD8+ T cells, and CAR expression was confirmed by flow cytometry. CAR33VH, similar to My96CAR, demonstrated robust and specific cytotoxicity in short-term and long-term co-incubation killing assays against CD33+ AML lines. In overnight cytokine release assays in which CAR T cells were challenged with the CD33+ tumor cells HL-60, MOLM-14 and KG-1a, CAR33VH elicited IFN-gamma, TNF-alpha and IL-2. This was seen with CD33+ cell lines, but not when CAR T were cultured alone. Studies with a CD33− cell line engineered to stably express the full length CD33 variant 1, or the naturally occurring CD33 splice variant 2, revealed that both CAR33VH and My96CAR, target the V domain of CD33, suggesting a similar therapeutic profile. Colony-formation assays utilizing peripheral blood CD34+ hematopoietic stem cells treated with CAR33VH, My96CAR, or with an untransduced T cell control, yielded similar numbers of BFU-E erythroid and CFU-GM myeloid colonies, suggesting a lack of CAR-related overt toxicity. In an in vivo AML model, NSG mice engrafted with MOLM-14 cells stably expressing firefly luciferase, both CAR33VH and CARMy96 efficiently eliminated tumors. In conclusion, we demonstrate for the first time the feasibility and efficacy of employing human variable domain-only binder derived from a phage display library in an anti-AML CAR design. CAR33VH, comprised of a human heavy-chain variable fragment-only antigen binding domain, was efficient in tumor killing in vitro and in vivo, and showed comparable functionality to the scFv-based My96CAR.

【 授权许可】

Unknown