Abstract
BackgroundClinical success with chimeric antigen receptor (CAR)- based immunotherapy for leukemia has been accompanied by the associated finding that antigen-escape variants of the disease are responsible for relapse. To target hematologic malignancies with a chimeric antigen receptor (CAR) that targets two antigens with a single vector, and thus potentially lessen the chance of leukemic escape mutations, a tandem-CAR approach was investigated.MethodsAntigen binding domains from the FMC63 (anti-CD19) and Leu16 (anti-CD20) antibodies were linked in differing configurations to transmembrane and T cell signaling domains to create tandem-CARs. Expression on the surface of primary human T cells was induced by transduction with a single lentiviral vector (LV) encoding the tandem-CAR. Tandem-CARs were compared to single antigen targeting CARs in vitro and in vivo, and to an admixture of transduced cells expressing each CAR in vivo in immunodeficient (NSG) disease-bearing mice.ResultsTandem constructs efficient killed the Raji leukemia cell line both in vitro and in vivo. Tandem CARs generated less cytokine than the CD20 CAR, but similar to CD19 CARs, on their own. In co-culture experiments at low effector to target ratios with both single- and tandem- CAR-T cells, a rapid down-modulation of full-length CD19 expression was seen on leukemia targets. There also was a partial down-modulation of CD22, and to a lesser degree, of CD20. Our data also highlight the extreme sensitivity of the NALM-6 cell line to general lymphocyte-mediated cytotoxicity. While single and tandem constructs were effective in vivo in a standard setting, in a high-disease burden setting, the tandem CAR proved both effective and less toxic than an admixture of transduced T cell populations expressing single CARs.ConclusionTandem CARs are equally effective in standard disease models to single antigen specificity CARs, and may be both more effective and less toxic in a higher disease burden setting. This may be due to optimized cell killing with more moderate cytokine production. The rapid co-modulation of CD19, CD20, and CD22 may account for the ability to rapidly evolve escape mutants by selecting for leukemic clones that not require these target antigens for continued expansion.
Highlights
Clinical success with chimeric antigen receptor (CAR)- based immunotherapy for leukemia has been accompanied by the associated finding that antigen-escape variants of the disease are responsible for relapse
An Single chain variable fragment (scFv) domain created from the Variable heavy chain domain (VH) and Variable light chain domain (VL) domains of an antiCD19 or anti-CD20 antibody can be linked to transmembrane sequences derived from CD28 or CD8, and linked to the intracellular signaling domains derived from the CD3-zeta chain and CD28 or CD137 [2, 3]
To study the effectiveness of tandem CD19 and CD20 targeting CARs, sequences encoding the antigen binding domains from the murine antibodies FMC63 and Leu16 were linked with a (GGGGS)5 sequence
Summary
Clinical success with chimeric antigen receptor (CAR)- based immunotherapy for leukemia has been accompanied by the associated finding that antigen-escape variants of the disease are responsible for relapse. One common approach to creating a cell population for adoptive immunotherapy is to isolate T cells by apheresis from the patient and to transduce these cells ex vivo with retroviral or lentiviral vectors that integrate into the host genome and express a chimeric antigen receptor (CAR), reviewed in [1]. The CAR confers both a binding domain derived from the scFv and the linked signaling domains in a single transmembrane protein that allows activation of a vector-transduced T cells. This transduced T cell population (CAR-T) can functionally target cells bearing the cognate antigen for destruction by active cytolysis, and by indirect immune effector mechanisms marshaled by the production of cytokines, such as interferon-gamma (IFNγ), interleukin-2 (IL-2), and tumor necrosis factor-alpha (TNFα). The effectiveness of CAR-modified T cells in adult hematologic malignancies has been more heterogeneous
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