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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
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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
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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.
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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.
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