Enhancement of antitumor activity by using a fully human gene encoding a single-chain fragmented antibody specific for carcinoembryonic antigen

Figures & data

Figure 1 Functional expression of CAR in Jurkat cells.

Notes: (A) Schematic representation of the CR gene within a mammalian expression vector. (B) A series of mCR genes with or without a hinge domain (CD8α or CD7) and a CD28 intracellular domain. The mouse scFv gene was combined directly with the intracellular domain of CD3ζ (mCR-0) or with a spacer (mCR-1 and mCR-3). The mCR-2 has CD28 between CD8α and CD3ζ. (C) The functional and specific binding of a series of mCR to CEA on transfected Jurkat cells were monitored by flow cytometry. Histograms are representative of Jurkat cells transfected with a series of mCR constructs using APC-BSA (left) or APC-CEA (right). Green and black lines on the left represent Jurkat cells with or without mCR-2, respectively. Jurkat cells without mCR-2 could not bind to BSA. Jurkat cells transfected with a series of mCRs could functionally bind to APC-CEA (right).
Abbreviations: APC, allophycocyanin; BSA, bovine serum albumin; CAR, chimeric antigen receptor; CEA, carcinoembryonic antigen; w/o, without.

Figure 2 The scFv-IL2 fusion protein maintains the functions of both component proteins.

Notes: (A) Schematic representation of the scFv-IL2 gene within the pBAD/gIII expression vector. The 45κHscFv and hIL-2 genes were combined by SOE-PCR. The scFv-IL2 gene was inserted into an Escherichia coli. expression vector. (B) Detection of scFv-IL2 using CBB staining and Western blotting. The scFv-IL2 and the parental 45κHscFv vectors were transformed into TOP10 cells and were expressed by the addition of D-arabinose. After concentration through an Ni⁺ column, the proteins were detected by CBB staining (lanes 1 and 3) and Western blotting using specific antibodies (lanes 1 and 3, anti-His; lane 5, anti-hIL-2; lane 6, anti-c-myc antibodies), at the expected sizes (28 and 45 kbp, respectively). (C) scFv-IL2 was detected by sandwich ELISA using rabbit and mouse anti-hIL-2 antibodies. Various concentrations of scFv-IL2 were incubated in rabbit anti-hIL-2 antibody-coated 96-well microtiter plates, and were then incubated with the mouse anti-hIL-2 antibody, followed by an HRP-conjugated goat anti-mouse antibody. After reacting with the OPD substrate, the absorption at 490 nm was determined using a plate reader. The absorption of scFv-IL2 wells increased in a dose-dependent manner. (D) The biological function of IL-2 in the scFv-IL2 fusion protein was determined using a cell proliferation assay. CTLL-2 cells were incubated with various concentrations of IL-2 and the equivalent scFv-IL2. After 24 h incubation, the cells were detected by WST-8 assay. CTLL-2 cells were proliferated by adding scFv-IL2 and IL-2. (E and F) The biological function of scFv antibody in the scFv-IL2 treated cells was detected by flow cytometry. Schematic representation of functional analysis of scFv antibody in scFv-IL2-treated cells. scFv-IL2 creates a bridge between MKN-45 CEA-positive cells and FITC-labeled anti-hIL-2 antibody (E). The fluorescence intensity of FITC was shifted to the right in the presence of scFv-IL2, compared with IL-2 (F).
Abbreviations: CBB, Coomassie Brilliant Blue; CEA, carcinoembryonic antigen; FITC, fluorescein isothiocyanate; OPD, o-Phenylenediamine-2HCl; scFv, single-chain fragmented antibody; SOE, splice-overlap extension.

Table 1 The sequences of specific primers used for SOE-PCR of scFv-IL2

Name	Sequences
45k-Fw	5′-CCGCGGTGGCGCGCCTTTGATCTC-3′
45H-RevL	5′-GGAACCACCCCCGCCTGTACCTCCGCCCCCAGACCCGCCTCCACCAGGCCCAACCGGCCA-3′
IL-2-FwL	5′-GGTGGAGGCGGGTCTGGGGGCGGATCAGGCGGGGGTGGTTCCATGGGTGCACCTACT-3′
IL-2-Rev	5′-AAAGCTTCGAGTCAGTGTTGA-3′

Abbreviations: SOE-PCR, splice-overlap extension polymerase chain reaction; scFv, single-chain fragmented antibody.

Figure 3 T cells transfected with mCR-2 were most effectively activated by MKN-45 cells.

Notes: (A) T-cell transfectants equivalently expressed a functional series of mCR on their surface. Black and purple lines represent APC-BSA and APC-CEA, respectively. (B) Expression of IFN-γ by a series of T-cell transfectants was detected by optical microscope after 1 day of incubation with MKN-45 cells, using a specific antibody. Among a series of mCR, mCR-2 could stimulate T cells and express IFN-γ most effectively in T-cell transfectants. Scale bar is 100 µm. (C) T cells transfected with mCR-2 bound MKN-45 CEA⁺ cells (upper), but not the cells lacking the CR gene (bottom), as well as APC-CEA did.
Abbreviations: APC, allophycocyanin; BSA, bovine serum albumin; CAR, chimeric antigen receptor; CEA, carcinoembryonic antigen.

Figure 4 Combination of CAR-bearing PBMCs and scFv-IL2 enhances the antitumor effect on MKN-45 cells.

Notes: (A) Exchange of the mouse scFv gene for its human equivalent in the CAR gene construct. The mouse scFv of mCR-2, which was the most effective among 4 CAR constructs, was exchanged for the 45κHscFv, a human scFv antibody, and this fully human CAR gene was designated hCR-2. (B) Expression of hCR-2 inserted into different expression vectors, pcDNA3.1(−) or pIRES, in Jurkat cells was detected by flow cytometry using APC-BSA and APC-CEA. Although hCR-2 was expressed at slightly higher levels in Jurkat cells than mCR-2, no other difference could be detected between the 2 expression vectors. (C) hCR-2 in PBMCs was detected by flow cytometry, by using EGFP expression. Approximately 60% of PBMCs expressed hCR-2 after transfection of the CAR gene within a pIRES vector using NEPA21. (D) PBMCs expressing hCR-2 in combination with scFv-IL2 demonstrated a higher antitumor activity on MKN-45 cells than those expressing IL-2 or PBMCs alone. Cell viability was determined by measuring the light products using a luciferase assay system. Data represent the mean ± standard error of the mean from at least 3 independent experiments. *P<0.05 or ^#P<0.05, significantly different from CAR-bearing PBMCs in combination with scFv-IL2 or the control (none) group, respectively.
Abbreviations: APC, allophycocyanin; BSA, bovine serum albumin; CAR, chimeric antigen receptor; CEA, carcinoembryonic antigen; PBMCs, peripheral blood mononuclear cells; scFv, single-chain fragmented antibody; w/o, without.

Figure S1 Artificial T-cell expansion (A) and the cell ratio of CD4⁺ and CD8⁺ T cells after 9-day expansion (B).

Figure S2 (A) Schematic representation of the combination of CAR-bearing PBMCs and scFv-IL2 for enhancing the antitumor activity to CEA⁺ tumor cells. (B) Conceptual representation of CAR-bearing PBMCs, DC, and scFv-IL2 fusion protein for enhancing the antitumor activity to cancer cells.

Abbreviations: CAR, chimeric antigen receptor; CEA, carcinoembryonic antigen; CTL, cytotoxic T lymphocytes; DC, dendric cells; NK, natural killer; PBMCs, peripheral blood mononuclear cells; scFv, single-chain fragmented antibody.

Table S1 Transfection efficiency of electroporation using Nucleofector™ and NEPA21

Cell type	Electroporation	Gene	Efficacy (%)	Cell viability (%)
Jurkat cells		EGFP	70	50
T cells	Nucleofector™	EGFP	50	60
T cells		CAR	37	>50
T cells		EGFP	>90	>90
PBMCs	NEPA21	EGFP	>60	95
PBMCs		CAR	>60	93

Abbreviations: CAR, chimeric antigen receptor; PBMCs, peripheral blood mononuclear cells.