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Liu et al. Supplementary Materials and Methods Cell Lines Hep G2, SK-HEP-1, HeLa, Hep 3B, Raji, Jurkat, Daudi, K562, Malme-3M, CA46, THP1, COLO-205, CaSki, MCF7, ASPC1, OVCAR3, LNCaP, A498, HEK293, A204, HUVEC and T2 (TAP-deficient cell line) were obtained from ATCC (Manassas, Virginia). Hep G2-Luc2 cells were purchased from Caliper (Alameda, California). All cells were cultured in RPMI 1640 or DMEM supplemented with 10% FCS. Cell lines used for in vivo experiments were tested for mycoplasma and authenticated by IDEXX BioResearch (Columbia, MO). HLA type and AFP expression was verified in house. Antibodies APC-conjugated anti-human CD3 (clone UCHT1), CD4 (clone RPA-T4) and CD8 (clone RPA-T8) were purchased from Biolegend. Anti-human CD107a (clone H4A3) and APCconjugated TNFα (clone MAb11), IFN-γ (clone 4S.B3), IL-2 (clone MQ1-17H12) and IL-6 (clone MQ2-13A5), PerCP-CCR7 (clone G043H7) and FITC-CD45RA (clone HI100) were purchased from BD Biosciences (San Jose, California). Phage panning A collection of 15 fully human naïve scFv and 8 semi-synthetic scFv antibody phage display libraries (diversity = 9x1010) constructed by Eureka Therapeutics (E-ALPHATM Phage Display) was used for the selection of human mAbs specific to AFP158/HLA-A*02:01 by cell panning as previously described (24), which was repeated three times. Positive clones were further tested via ELISA using plates coated with AFP158/HLA-A*02:01 complexes or control 1 Liu et al. peptide/HLA-A*02:01 complexes. Binding of phage clones was detected by HRP-conjugated anti-M13 antibody and reading absorbance at 450nm. Generation of full-length IgG1 antibodies The antibody variable regions were sub-cloned into mammalian expression vectors with mouse IgG1 heavy and light chain constant regions and expressed in HEK293 cells as previously described (24). The binding affinity of the mouse chimeric IgG1 antibodies towards AFP158/HLA-A*02:01 complexes was determined using Surface Plasma Resonance (BIACore X100, GE Healthcare), according to the manufacturer’s protocol for single-cycle kinetics measurement. BsAb and CAR T cell killing assay BsAbs were generated using the phage clone scFv sequence at the N-terminal and a mouse-anti-human CD3ε at the C-terminal (43). For BsAb killing assays, activated T cells and target cells were co-cultured at an effector-to-target cell ratio (E:T) of 5:1 with different concentrations of BsAbs for 16 hours. The BsAb killing assay was repeated three times. For CAR T cell killing assays, CAR+ T cells and target cells were co-cultured at an effector-to-target cell ratio (E:T) of 5:1 for 16 hours. For killing assays involving pulsed recombinant human AFP, target cells were incubated overnight with human AFP (0.4µg/mL) prior to killing assay. Cytotoxicity was determined using a lactate dehydrogenase (LDH) release assay (Promega). Cytokine and degranulation assays Cytokine release was measured using the Magpix multiplex system (Luminex, Austin, Texas) with the Bio-plex Pro Human Cytokine 8-plex Assays (BioRad, Hercules, California). Intracellular staining of cytokines was conducted after 4 hours of co-culturing target cells with 2 Liu et al. mock or AFP-CAR T cells in the presence of a protein transport inhibitor cocktail (eBiosicences, San Diego, California). CD8+ cells were inferred from the CD4- population. For the degranulation assay, AFP-CAR T cells were co-incubated with target cells for 4 hours in the presence of 1:200 anti-CD107a and protein transport inhibitor cocktail (eBioscience) then assayed by flow cytometry to detect CD107a. Matrigel infiltration 24-well plates were coated with 250 µl of Matrigel (Corning) per well and incubated at 37°C for 30 minutes to solidify. 250,000 cells were added to each well and the plate was incubated for another 30 minutes. The medium was then replaced with an additional 300 µl of Matrigel per well, and after solidifying, 1 mL of growth medium was added to each well. Matrigel-embedded cells were cultured for 6-8 days to form cell clusters, after which 1x106 CAR T cells were added. After a 2-day incubation, infiltration of T cells was confirmed by visual inspection; propidium iodide was added to a final concentration of 2 µg/mL. Phase contrast and fluorescent images were captured at the focal plane of clustered cells. This experiment was done twice. Assessment of T cell subsets Eleven days post-transduction, CAR or mock T cells were co-stained with PE-Tetramer, APC-CD8, PerCP-CCR7 and FITC-CD45RA and analyzed using flow cytometry. CD4+ cells were inferred from the CD8- population. T cells were consistently >99% CD3+, with >99% of the cells being either CD4+ or CD8+, enabling us to infer CD4+ from CD8- and vice versa. 3 Liu et al. Mouse xenograft models of liver cancer All animal experiments were conducted according to protocols approved by their Institutional Animal Care and Use Committee (IACUC) and in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, National Academy Press, Washington, DC, 1996) and the Policy on Humane Care and Use of Laboratory Animals (Department of Health and Human Services, Bethesda, MD). Animal handlers were blinded to the nature of treatment and identification of treatment arms. The sample size for animal studies was determined based on the variation in tumor growth associated with each model. The number of mice was determined to meet adequate statistical significance based on previous experience and/or published work. For the Hep G2 i.p. and NSG studies, 6 mice per group were selected to gather preliminary data for proof-of-concept in these models. Randomization into treatment arms was done such that the average and standard deviation of tumor burden/size were equal in all groups. Determination of AFP158/HLA-A*02:01 complex number on cell surfaces A Quantum™ Simply Cellular® anti-Mouse IgG kit (Bangs Laboratories) was used to quantify the AFP158/HLA-A*02:01 complex number on the surface of cells using the manufacturer’s protocol. In brief, a standard curve correlating MFI to antigen binding capacity (ABC) was generated. T2 cells were loaded with varying amounts of AFP158 peptide in order to determine the limit of detection of this assay for ET1402L1 chimeric mouse IgG. Lastly, SKHEP-1, SK-HEP-1-MG, and Hep G2 cells were analyzed to quantify the ABC. 4