TY - JOUR T1 - A tandem CD19/CD20 CAR lentiviral vector drives on-target and off-target antigen modulation in leukemia cell lines JF - Journal for ImmunoTherapy of Cancer JO - J Immunother Cancer DO - 10.1186/s40425-017-0246-1 VL - 5 IS - 1 SP - 42 AU - Dina Schneider AU - Ying Xiong AU - Darong Wu AU - Volker Nӧlle AU - Sarah Schmitz AU - Waleed Haso AU - Andrew Kaiser AU - Boro Dropulic AU - Rimas J. Orentas Y1 - 2017/12/01 UR - http://jitc.bmj.com/content/5/1/42.abstract N2 - Background 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. 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.Methods Antigen 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.Results Tandem 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.Conclusion Tandem 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.Abbreviations:7AADSeven-actinomycin DAAAmino acidsAFAlexa fluorALLAcute lymphoblastic leukemiaANOVAAnalysis of varianceAPCAllophycocyaninCAR TChimeric antigen receptor T cellsCDCluster of differentiationCLLChronic lymphocytic leukemiaCPSCounts per secondCTLCytotoxic T lymphocyteELISAEnzyme linked immunosorbent assayFBSFetal bovine serumFcFragment crystallizable regionFITCFluorescein isothiocyanateGFPGreen fluorescent proteinGM-CSFGranulocyte macrophage colony stimulating factorGMPGood manufacturing practiceHCLHairy cell leukemiaHEKHuman embryonic kidney cellsHer2Human epidermal growth factor receptor twoIFNγInterferon gammaIL-2Interleukin twoIUInternational unitsLucFirefly luciferaseLVLentiviral vectorMCLMantle cell lymphomaMOPSThree-(N-morpholino)propanesulfonic acidN.T.Non-transduced T cells control from same donorNSGNOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJOBIOklahoma Blood InstitutePEPhycoerythrinPLLProlymphocytic leukemiaROR1Receptor tyrosine kinase-like orphan receptor 1RPMRevolutions per minutescFvSingle chain variable fragmentSDS-PAGESodium dodecyl sulfate polyacrylamide gel electrophoresisSLVLSplenic lymphoma with villous lymphocytesT.A.Tumor alone control groupTNFαTumor necrosis factor alphaVHVariable heavy chain domainVLVariable light chain domain ER -