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Advances in Engineered T Cells

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Front. Oncol. | doi: 10.3389/fonc.2018.00539

A unique human immunoglobulin heavy chain variable domain-only CD33 CAR for the treatment of acute myeloid leukemia

 Dina Schneider1*, Ying Xiong1, Peirong Hu1, Darong Wu1,  Weizao Chen2,  Tianlei Ying2, 3, Zhongyu Zhu1, 2, Dimiter S. Dimitrov2, 4,  Boro Dropulic1 and  Rimas J. Orentas1, 5
  • 1Lentigen Technology (United States), United States
  • 2Cancer and Inflammation Program, Center for Cancer Research (NCI), United States
  • 3Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Fudan University, China
  • 4Center for Antibody Therapeutics, Department of Medicine, School of Medicine, University of Pittsburgh, United States
  • 5Seattle Children's Research Institute, United States

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, similarly 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.

Keywords: lentiviral (LV) vector, CD33 antigen, CAR T cell, Immunotherapy, AML - acute myeloid leukemia

Received: 22 Jul 2018; Accepted: 01 Nov 2018.

Edited by:

Peter Bader, Universitätsklinikum Frankfurt, Germany

Reviewed by:

Evelyn Ullrich, Goethe-Universität Frankfurt am Main, Germany
Halvard Bonig, German Red Cross Blood Service and Institute for Transfusion Medicine and Immunohematology of the KGU, Frankfurt, Germany  

Copyright: © 2018 Schneider, Xiong, Hu, Wu, Chen, Ying, Zhu, Dimitrov, Dropulic and Orentas. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: PhD. Dina Schneider, Lentigen Technology (United States), Gaithersburg, United States,