doi: 10.1038/s41408-018-0113-4.
Development of a novel target module redirecting UniCAR T cells to Sialyl Tn-expressing tumor cells
Affiliations
- PMID: 30190468
- PMCID: PMC6127150
- DOI: 10.1038/s41408-018-0113-4
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Development of a novel target module redirecting UniCAR T cells to Sialyl Tn-expressing tumor cells
Blood Cancer J.
.
No abstract available
Conflict of interest statement
L.R.L., C.N., M.B., and P.V. have filed patents related to anti-STn mAb L2A5. M.B. has filed patents related to the UniCAR system. M.B. is shareholder of the company GEMoaB which owns the IP related to the UniCAR system. The remaining authors declare that they have no conflict of interest.
Figures
a The UniCAR system consists of T cells genetically engineered with UniCARs, containing a humanized scFv derived from the anti-La mAb E5B9 fused to the transmembrane domain of human CD28. Intracellularly, the construct comprises signaling domains of CD28 and CD3ζ. In the absence of an antigen-specific TM, UniCAR T cells are unable to bind to target cells. However, in the presence of an anti-STn TM a complex is formed via the UniCAR epitope (E5B9), endorsing binding of UniCAR T cells to STn-expressing cancer cells. b The anti-STn TM comprises the variable domains derived from the mAb L2A5 arranged in VH–VL orientation separated by a (G4S)3 peptide linker. The UniCAR epitope was fused C-terminally and is flanked by two spacer peptides (N-terminal spacer: AAA; C-terminal spacer: G4S). To allow protein purification and detection, the TM was tagged with 6xhis residues at the C-terminus. In addition, an N-terminal leader peptide (LP) was added to ensure secretion of the recombinant protein into the cell culture supernatant. c T cell-mediated tumor cell killing was measured using standard chromium release assays. MDA-MB-231 and MCR STn-expressing cell lines were incubated with T cells engrafted with either the vector control (vector backbone encoding only the EGFP marker protein), UniCAR STOP construct (lacking intracellular signaling domains) or UniCAR signaling construct (UniCAR 28/ζ). Both tumor cell lines were cultivated with the respective genetically engineered T cells at an effector to target (E:T) ratio of 5:1 in the presence or absence of 80 nM anti-STn TM for 24 h . Mean specific lysis and SD of three independent T cell donors are shown. d MDA-MB-231 STn-expressing cells were transduced to express firefly luciferase resulting in MDA-MB-231 STn-Luc cells. Per mouse, either 1.5 × 106 tumor cells alone, mixed with 1 × 106 UniCAR 28/ζ T cells or mixed with 1 × 106 UniCAR 28/ζ T cells and 10 µg of anti-STn TM were injected subcutaneously into female NMRI-Foxn1nu/Foxn1nu mice resulting in three groups of animals each consisting of five mice. Quantitative evaluation of the luminescence imaging of anesthetized mice at day 0 and followed at day 1, 3, and 6. e1,e2 Genetically engineered UniCAR T cells were incubated for 24 h in the presence or absence of MDA-MB-231 STn+ (e1) or MCR STn+ (e2) cells as well as in the presence or absence of 80 nM anti-STn TM. As controls, the same conditions were performed using T cells transduced with the vector control or with the UniCAR STOP. Cell culture supernatants from three individual donors were collected and analyzed using MACSPlex Cytokine 12 Kit and MACSQuantify® software according to the manufacturer’s instructions. SD of mean cytokine values of three independent T cell donors are shown. Statistical analysis was performed using one-way or two-way ANOVA with Bonferroni multiple-comparison test (ns = not significant; *p < 0.1; **p < 0.01; ***p < 0.001, and ****p < 0.0001)
[64Cu]Cu-NODAGA-anti-STn TM was developed according to standard procedures and intravenously injected in NMRI-Foxn1nu/nu MDA-MB-231 STn-Luc tumor-bearing mice with 3–4 weeks of tumor growth (1.5 × 106 cells, n = 4). Two hours after injection, the biodistribution of the [64Cu]Cu-NODAGA-anti-STn TM was measured and shown as activity concentration (SUV) (a) or percentage of the total activity of the injected dose %ID (b) in the selected organs and tissues. Target to background ratios including tumor to muscle-, and tumor to blood ratios are shown in (c). Values are represented as mean ± SEM. d, e Representative orthogonal images of a PET study at 1.5 h (d) and 13 h (e) after single intravenous injection of ~7 MBq [64Cu]Cu-NODAGA-anti-STn TM. f Time activity concentration curves of representative organs of an NMRI-Foxn1nu/nu mouse bearing an MDA-MB-231 STn-expressing tumor derived from a dynamic PET study over 2 h and a static at 13 h after single intravenous injection of ~7 MBq [64Cu]Cu-NODAGA-anti-STn TM. g Resulting target to background curves calculated as tumor to muscle and tumor to blood ratios
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