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. 2017 Aug 22;10:3979-3990.
doi: 10.2147/OTT.S140174. eCollection 2017.

Enhancement of Antitumor Activity by Using a Fully Human Gene Encoding a Single-Chain Fragmented Antibody Specific for Carcinoembryonic Antigen

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Free PMC article

Enhancement of Antitumor Activity by Using a Fully Human Gene Encoding a Single-Chain Fragmented Antibody Specific for Carcinoembryonic Antigen

Hirotomo Shibaguchi et al. Onco Targets Ther. .
Free PMC article

Abstract

Human leukocyte antigen and/or costimulatory molecules are frequently lacking in metastatic tumor cells, and thus tumor cells are able to escape from the immune system. Although lymphocytes with a chimeric antigen receptor (CAR) is a promising approach for overcoming this challenge in cancer immunotherapy, administration of modified T cells alone often demonstrates little efficacy in patients. Therefore, in order to enhance the antitumor activity of immune cells in the cancer microenvironment, we used lymphocytes expressing CAR in combination with a fusion protein of IL-2 that contained the single-chain fragmented antibody (scFv) specific for the carcinoembryonic antigen. Among a series of CAR constructs, with or without a spacer and the intracellular domain of CD28, the CAR construct containing CD8α, CD28, and CD3ζ most effectively activated and expressed INF-γ in CAR-bearing T cells. Furthermore, in comparison with free IL-2, the combination of peripheral blood mononuclear cells expressing CAR and the fusion protein containing IL-2 significantly enhanced the antitumor activity against MKN-45 cells, a human gastric cancer cell line. In conclusion, this novel combination therapy of CAR and a fusion protein consisting of a functional cytokine and a fully human scFv may be a promising approach for adoptive cancer immunotherapy.

Keywords: CEA; chimeric antigen receptor; combination therapy; fusion protein; human scFv.

Conflict of interest statement

Disclosure The authors report no conflicts of interest in this work.

Figures

Figure 1
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
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.
Figure 3
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
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.

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