The online version of this article (doi:10.1186/s13045-017-0437-8) contains supplementary material, which is available to authorized users.
Multiple iterations of chimeric antigen receptors (CARs) have been developed, mainly focusing on intracellular signaling modules. However, the effect of non-signaling extracellular modules on the expansion and therapeutic efficacy of CARs remains largely undefined.
We generated two versions of CAR vectors, with or without a hinge domain, targeting CD19, mesothelin, PSCA, MUC1, and HER2, respectively. Then, we systematically compared the effect of the hinge domains on the growth kinetics, cytokine production, and cytotoxicity of CAR T cells in vitro and in vivo.
During in vitro culture period, the percentages and absolute numbers of T cells expressing the CARs containing a hinge domain continuously increased, mainly through the promotion of CD4+ CAR T cell expansion, regardless of the single-chain variable fragment (scFv). In vitro migration assay showed that the hinges enhanced CAR T cells migratory capacity. The T cells expressing anti-CD19 CARs with or without a hinge had similar antitumor capacities in vivo, whereas the T cells expressing anti-mesothelin CARs containing a hinge domain showed enhanced antitumor activities.
Hence, our results demonstrate that a hinge contributes to CAR T cell expansion and is capable of increasing the antitumor efficacy of some specific CAR T cells. Our results suggest potential novel strategies in CAR vector design.
Additional file 1: Figure S1. Hinge incorporation can promote the expansion of CAR T cells. Flow cytometric analysis of the percentage of 19.28z, 19-H.28z T cells, Meso.28z, Meso-H.28z T cells, PSCA.28z, PSCA-H.28z T cells, and GFP control T cells from day 6 to 15 during the in vitro culture period. The data are representative of independent experiments verified with cells from over three individual healthy human donors. (JPG 2876 kb)
Additional file 2: Figure S2. Hinge incorporation promotes CD4+ anti-CD19 CAR T cell expansion. Flow cytometric analysis of the percentage of CD4+ and CD8+ GFP T, 19.28z T, and 19-H.28z T during the in vitro culture period. The data are representative of independent experiments verified with cells from over three individual healthy human donors. (JPG 2320 kb)
Additional file 3: Figure S3. Percentages of CD4+ and CD8+ CAR T cells without a hinge domain both tended to be stable throughout the in vitro culture period. Flow cytometric analysis of the percentage of CD4+ and CD8+ (A) 19.28z T, (B) Meso.28z T, (C) PSCA.28z T, (D) HER2.28z T, and (E) GFP control T cells during the in vitro culture period. The data are representative of independent experiments verified with cells from over three individual healthy human donors. (JPG 924 kb)
Cai B, Guo M, Wang Y, Zhang Y, Yang J, Guo Y, Dai H, Yu C, Sun Q, Qiao J, et al. Co-infusion of haplo-identical CD19-chimeric antigen receptor T cells and stem cells achieved full donor engraftment in refractory acute lymphoblastic leukemia. J Hematol Oncol. 2016;9(1):131. CrossRefPubMedPubMedCentral
Kowolik CM, Topp MS, Gonzalez S, Pfeiffer T, Olivares S, Gonzalez N, Smith DD, Forman SJ, Jensen MC, Cooper LJ. CD28 costimulation provided through a CD19-specific chimeric antigen receptor enhances in vivo persistence and antitumor efficacy of adoptively transferred T cells. Cancer Res. 2006;66(22):10995–1004. CrossRefPubMed
Milone MC, Fish JD, Carpenito C, Carroll RG, Binder GK, Teachey D, Samanta M, Lakhal M, Gloss B, Danet-Desnoyers G, et al. Chimeric receptors containing CD137 signal transduction domains mediate enhanced survival of T cells and increased antileukemic efficacy in vivo. Mol Ther. 2009;17(8):1453–64. CrossRefPubMedPubMedCentral
Guest RD, Hawkins RE, Kirillova N, Cheadle EJ, Arnold J, O'Neill A, Irlam J, Chester KA, Kemshead JT, Shaw DM, et al. The role of extracellular spacer regions in the optimal design of chimeric immune receptors: evaluation of four different scFvs and antigens. J Immunother. 2005;28(3):203–11. CrossRefPubMed
Moritz D, Groner B. A spacer region between the single chain antibody- and the CD3 zeta-chain domain of chimeric T cell receptor components is required for efficient ligand binding and signaling activity. Gene Ther. 1995;2(8):539–46. PubMed
Hombach A, Hombach AA, Abken H. Adoptive immunotherapy with genetically engineered T cells: modification of the IgG1 Fc ‘spacer’ domain in the extracellular moiety of chimeric antigen receptors avoids ‘off-target’ activation and unintended initiation of an innate immune response. Gene Ther. 2010;17(10):1206–13. CrossRefPubMed
Yuan J, Kashiwagi S, Reeves P, Nezivar J, Yang Y, Arrifin NH, Nguyen M, Jean-Mary G, Tong X, Uppal P, et al. A novel mycobacterial Hsp70-containing fusion protein targeting mesothelin augments antitumor immunity and prolongs survival in murine models of ovarian cancer and mesothelioma. J Hematol Oncol. 2014;7:15. CrossRefPubMedPubMedCentral
You F, Jiang L, Zhang B, Lu Q, Zhou Q, Liao X, Wu H, Du K, Zhu Y, Meng H, et al. Phase 1 clinical trial demonstrated that MUC1 positive metastatic seminal vesicle cancer can be effectively eradicated by modified Anti-MUC1 chimeric antigen receptor transduced T cells. Sci China Life Sci. 2016;59(4):386–97. CrossRefPubMed
Ye W, Jiang Z, Lu X, Ren X, Deng M, Lin S, Xiao Y, Lin S, Wang S, Li B et al. GZD824 suppresses the growth of human B cell precursor acute lymphoblastic leukemia cells by inhibiting the SRC kinase and PI3K/AKT pathways. Oncotarget. 2016. doi: 10.18632/oncotarget.10881.
Dai H, Wang Y, Lu X, Han W. Chimeric Antigen Receptors Modified T-Cells for Cancer Therapy. J Natl Cancer Inst. 2016;108(7):djv439.
Geyer MB, Brentjens RJ. Review: Current clinical applications of chimeric antigen receptor (CAR) modified T cells. Cytotherapy. 2016;18(11):1393-409.
Turtle CJ, Hanafi LA, Berger C, Hudecek M, Pender B, Robinson E, Hawkins R, Chaney C, Cherian S, Chen X, et al. Immunotherapy of non-Hodgkin’s lymphoma with a defined ratio of CD8+ and CD4+ CD19-specific chimeric antigen receptor-modified T cells. Sci Transl Med. 2016;8(355):355ra116. CrossRefPubMedPubMedCentral
Golubovskaya V, Wu L. Different Subsets of T Cells, Memory, Effector Functions, and CAR-T Immunotherapy. Cancers. 2016;8(3):36.
Huls MH, Figliola MJ, Dawson MJ, Olivares S, Kebriaei P, Shpall EJ, Champlin RE, Singh H, Cooper LJ. Clinical application of Sleeping Beauty and artificial antigen presenting cells to genetically modify T cells from peripheral and umbilical cord blood. J Vis Exp. 2013;72:e50070.
- Incorporation of a hinge domain improves the expansion of chimeric antigen receptor T cells
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