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. 2006;2006(2):68091.
doi: 10.1155/JBB/2006/68091.

Cloning and expression of human membrane-bound and soluble engineered T cell receptors for immunotherapy

Nehad M Alajez  1 , Saman Eghtesad, Olivera J Finn
Affiliations

Cloning and expression of human membrane-bound and soluble engineered T cell receptors for immunotherapy

Nehad M Alajez et al. J Biomed Biotechnol. 2006.

Abstract

We report here the design and construction of several gene vectors for expression in mammalian cells of membrane-bound and soluble human T cell receptors (TR). We designed a vector (TR-ALPHA-IRES-TR-BETA pEF4) that encodes high-level expression of the full-length TR on the surface of T cells. Furthermore, we engineered TR that does not require the presence of endogenous CD3 molecules for surface expression and thus expression is not limited to T cells. We also constructed a vector encoding a single-chain TR (scTR) as a fusion protein of V-ALPHA-V-BETA-C-BETA with CD3Z. Since it is encoded and expressed as a single molecule, this scTR is well suited for gene therapy. Lastly, we successfully used a mammalian expression vector for generation of soluble human TR. The approaches we used here for manipulation of a human tumor-specific TR can be useful for other investigators interested in TR-based immunotherapy.

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Figures

Figure 1
Figure 1
Expression of the TRAZ from MA CTL clone on the surface of a TR-deficient Jurkat line (JRT3-T3.5). (a) The TR-ALPHA-IRES-TR-BETA cassette was cloned into the pEF4 expression vector. (b) Untransfected JRT3-T3.5, (c) JRT3-T3.5 cells transfected with the TCRB sequence, or (d) JRT3-T3.5 cells transfected with MA TR-ALPHA-IRES-TR-BETA pEF4 were stained with anti-CD3 Epsilon (open histogram) or with isotype control (filled histogram). IRES stands for internal ribosomal entry site. Zeocin is antibiotic resistance gene.
Figure 1
Figure 1
Expression of the TRAZ from MA CTL clone on the surface of a TR-deficient Jurkat line (JRT3-T3.5). (a) The TR-ALPHA-IRES-TR-BETA cassette was cloned into the pEF4 expression vector. (b) Untransfected JRT3-T3.5, (c) JRT3-T3.5 cells transfected with the TCRB sequence, or (d) JRT3-T3.5 cells transfected with MA TR-ALPHA-IRES-TR-BETA pEF4 were stained with anti-CD3 Epsilon (open histogram) or with isotype control (filled histogram). IRES stands for internal ribosomal entry site. Zeocin is antibiotic resistance gene.
Figure 1
Figure 1
Expression of the TRAZ from MA CTL clone on the surface of a TR-deficient Jurkat line (JRT3-T3.5). (a) The TR-ALPHA-IRES-TR-BETA cassette was cloned into the pEF4 expression vector. (b) Untransfected JRT3-T3.5, (c) JRT3-T3.5 cells transfected with the TCRB sequence, or (d) JRT3-T3.5 cells transfected with MA TR-ALPHA-IRES-TR-BETA pEF4 were stained with anti-CD3 Epsilon (open histogram) or with isotype control (filled histogram). IRES stands for internal ribosomal entry site. Zeocin is antibiotic resistance gene.
Figure 1
Figure 1
Expression of the TRAZ from MA CTL clone on the surface of a TR-deficient Jurkat line (JRT3-T3.5). (a) The TR-ALPHA-IRES-TR-BETA cassette was cloned into the pEF4 expression vector. (b) Untransfected JRT3-T3.5, (c) JRT3-T3.5 cells transfected with the TCRB sequence, or (d) JRT3-T3.5 cells transfected with MA TR-ALPHA-IRES-TR-BETA pEF4 were stained with anti-CD3 Epsilon (open histogram) or with isotype control (filled histogram). IRES stands for internal ribosomal entry site. Zeocin is antibiotic resistance gene.
Figure 2
Figure 2
Construction and expression of engineered MUC1-specific TRAZ and TRBZ receptors. Expression vectors for (a) TRAZ, (b) BZ, and (c) AZ/BZ. (d) Untransfected 293H cells, (e) 293H cells cotransfected with the TRAZ and TRBZ, or (f) cells transfected with the AZ-IRES-BZ pLNCX2 were stained for surface expression of the TR using anti-TR antibody BF1 (open histogram) or isotype control antibody (filled histogram).
Figure 2
Figure 2
Construction and expression of engineered MUC1-specific TRAZ and TRBZ receptors. Expression vectors for (a) TRAZ, (b) BZ, and (c) AZ/BZ. (d) Untransfected 293H cells, (e) 293H cells cotransfected with the TRAZ and TRBZ, or (f) cells transfected with the AZ-IRES-BZ pLNCX2 were stained for surface expression of the TR using anti-TR antibody BF1 (open histogram) or isotype control antibody (filled histogram).
Figure 2
Figure 2
Construction and expression of engineered MUC1-specific TRAZ and TRBZ receptors. Expression vectors for (a) TRAZ, (b) BZ, and (c) AZ/BZ. (d) Untransfected 293H cells, (e) 293H cells cotransfected with the TRAZ and TRBZ, or (f) cells transfected with the AZ-IRES-BZ pLNCX2 were stained for surface expression of the TR using anti-TR antibody BF1 (open histogram) or isotype control antibody (filled histogram).
Figure 2
Figure 2
Construction and expression of engineered MUC1-specific TRAZ and TRBZ receptors. Expression vectors for (a) TRAZ, (b) BZ, and (c) AZ/BZ. (d) Untransfected 293H cells, (e) 293H cells cotransfected with the TRAZ and TRBZ, or (f) cells transfected with the AZ-IRES-BZ pLNCX2 were stained for surface expression of the TR using anti-TR antibody BF1 (open histogram) or isotype control antibody (filled histogram).
Figure 2
Figure 2
Construction and expression of engineered MUC1-specific TRAZ and TRBZ receptors. Expression vectors for (a) TRAZ, (b) BZ, and (c) AZ/BZ. (d) Untransfected 293H cells, (e) 293H cells cotransfected with the TRAZ and TRBZ, or (f) cells transfected with the AZ-IRES-BZ pLNCX2 were stained for surface expression of the TR using anti-TR antibody BF1 (open histogram) or isotype control antibody (filled histogram).
Figure 2
Figure 2
Construction and expression of engineered MUC1-specific TRAZ and TRBZ receptors. Expression vectors for (a) TRAZ, (b) BZ, and (c) AZ/BZ. (d) Untransfected 293H cells, (e) 293H cells cotransfected with the TRAZ and TRBZ, or (f) cells transfected with the AZ-IRES-BZ pLNCX2 were stained for surface expression of the TR using anti-TR antibody BF1 (open histogram) or isotype control antibody (filled histogram).
Figure 3
Figure 3
Construction and expression of MUC1-specific single-chain T cell receptors (scTRs). (a) 293H cells were transfected with (b) the scTR, (c) scTR-CD4TM-hZ, or (d) scTR-CD4TM-AGD-hZ mammalian expression vectors. Cells were stained with anti-TR BF1 (open histogram) or isotype control (filled histogram) antibody. (e) shows quantitative comparison of TR expression on 293H cells transfected with different scTR constructs. P < .05.
Figure 3
Figure 3
Construction and expression of MUC1-specific single-chain T cell receptors (scTRs). (a) 293H cells were transfected with (b) the scTR, (c) scTR-CD4TM-hZ, or (d) scTR-CD4TM-AGD-hZ mammalian expression vectors. Cells were stained with anti-TR BF1 (open histogram) or isotype control (filled histogram) antibody. (e) shows quantitative comparison of TR expression on 293H cells transfected with different scTR constructs. P < .05.
Figure 3
Figure 3
Construction and expression of MUC1-specific single-chain T cell receptors (scTRs). (a) 293H cells were transfected with (b) the scTR, (c) scTR-CD4TM-hZ, or (d) scTR-CD4TM-AGD-hZ mammalian expression vectors. Cells were stained with anti-TR BF1 (open histogram) or isotype control (filled histogram) antibody. (e) shows quantitative comparison of TR expression on 293H cells transfected with different scTR constructs. P < .05.
Figure 3
Figure 3
Construction and expression of MUC1-specific single-chain T cell receptors (scTRs). (a) 293H cells were transfected with (b) the scTR, (c) scTR-CD4TM-hZ, or (d) scTR-CD4TM-AGD-hZ mammalian expression vectors. Cells were stained with anti-TR BF1 (open histogram) or isotype control (filled histogram) antibody. (e) shows quantitative comparison of TR expression on 293H cells transfected with different scTR constructs. P < .05.
Figure 3
Figure 3
Construction and expression of MUC1-specific single-chain T cell receptors (scTRs). (a) 293H cells were transfected with (b) the scTR, (c) scTR-CD4TM-hZ, or (d) scTR-CD4TM-AGD-hZ mammalian expression vectors. Cells were stained with anti-TR BF1 (open histogram) or isotype control (filled histogram) antibody. (e) shows quantitative comparison of TR expression on 293H cells transfected with different scTR constructs. P < .05.
Figure 4
Figure 4
Expression of functional scTR on the surface of T and non-T immune cells. (a) Rat basophilic leukemia (RBL) or BWZ cells were transfected with the scTR-pEF6 and were stained for surface expression with anti-TR BF1 antibody (open) or with isotype control (filled) histogram. (b) IL-2 secretion from BWZ cells (open bars) or BWZ-scTR (filled bars) following stimulated with SEE superantigen or with anti-TR BF1 antibody. Stimulation with Ionomycin/PMA (I/P) served as the positive control.
Figure 4
Figure 4
Expression of functional scTR on the surface of T and non-T immune cells. (a) Rat basophilic leukemia (RBL) or BWZ cells were transfected with the scTR-pEF6 and were stained for surface expression with anti-TR BF1 antibody (open) or with isotype control (filled) histogram. (b) IL-2 secretion from BWZ cells (open bars) or BWZ-scTR (filled bars) following stimulated with SEE superantigen or with anti-TR BF1 antibody. Stimulation with Ionomycin/PMA (I/P) served as the positive control.
Figure 5
Figure 5
Expression and purification of soluble scTR following surface biotin labeling and immunoprecipitation. (a) scTR expression vector encoding a thrombin cleavage site, T. (b) RBL cells transfected with the scTR were stained with anti-TR BF1 antibody (open histograms) before (right) or after (left) treatment with thrombin. Filled histogram shows staining with isotype control antibody. (c) Immunoprecipitation of the scTR from RBL (lanes 1 and 3) or RBL cells transfected with the scTR (lanes 2 and 4) before (lanes 1 and 2) or after (lanes 3 and 4) treatment with thrombin. Lanes 6–8 are SA-HRP blotting of fraction eluted with 150 mM Glycine, PH 2.2, 100 mM Glycine PH 2.2, or diethylamine (DEA) PH 11.2, respectively. Lane 5 is IP from control lysate.
Figure 5
Figure 5
Expression and purification of soluble scTR following surface biotin labeling and immunoprecipitation. (a) scTR expression vector encoding a thrombin cleavage site, T. (b) RBL cells transfected with the scTR were stained with anti-TR BF1 antibody (open histograms) before (right) or after (left) treatment with thrombin. Filled histogram shows staining with isotype control antibody. (c) Immunoprecipitation of the scTR from RBL (lanes 1 and 3) or RBL cells transfected with the scTR (lanes 2 and 4) before (lanes 1 and 2) or after (lanes 3 and 4) treatment with thrombin. Lanes 6–8 are SA-HRP blotting of fraction eluted with 150 mM Glycine, PH 2.2, 100 mM Glycine PH 2.2, or diethylamine (DEA) PH 11.2, respectively. Lane 5 is IP from control lysate.
Figure 5
Figure 5
Expression and purification of soluble scTR following surface biotin labeling and immunoprecipitation. (a) scTR expression vector encoding a thrombin cleavage site, T. (b) RBL cells transfected with the scTR were stained with anti-TR BF1 antibody (open histograms) before (right) or after (left) treatment with thrombin. Filled histogram shows staining with isotype control antibody. (c) Immunoprecipitation of the scTR from RBL (lanes 1 and 3) or RBL cells transfected with the scTR (lanes 2 and 4) before (lanes 1 and 2) or after (lanes 3 and 4) treatment with thrombin. Lanes 6–8 are SA-HRP blotting of fraction eluted with 150 mM Glycine, PH 2.2, 100 mM Glycine PH 2.2, or diethylamine (DEA) PH 11.2, respectively. Lane 5 is IP from control lysate.
Figure 6
Figure 6
Expression and purification of soluble scTR using mammalian expression system. (a) The single-chain fraction variable (scFv) domain was cloned and fused to a C-terminus HA and c-myc epitope tags. (b) Secreted scTCR that was cloned and fused to a C-terminus Flag and 6-His epitope tags. (c) and (d) the secreted scTR as described in (b), with the exception that the secreted scTR was fused to the leader sequence from GM-CSF (c) or from Ig-κ light chain (d). (e) Western blot of the culture supernatants from 293H cells transiently transfected with constructs a–d (a′–d′), immunoprecipitated with appropriate anti-tag antibody and blotted with anti-c-myc antibody (a′) or with anti-Flag-M2 antibody (b′–d′). Ctr is supernatant from untransfected cells. (f) Coomassie blue staining of fractions from culture supernatant b′ purified using nickel column. Lane 1 is culture supernatant before purification, 2 is flow through, 3 is wash, and 4–6 are different eluted fractions. (g) Western blot of panel (f) using anti-Flag M2 antibody.
Figure 6
Figure 6
Expression and purification of soluble scTR using mammalian expression system. (a) The single-chain fraction variable (scFv) domain was cloned and fused to a C-terminus HA and c-myc epitope tags. (b) Secreted scTCR that was cloned and fused to a C-terminus Flag and 6-His epitope tags. (c) and (d) the secreted scTR as described in (b), with the exception that the secreted scTR was fused to the leader sequence from GM-CSF (c) or from Ig-κ light chain (d). (e) Western blot of the culture supernatants from 293H cells transiently transfected with constructs a–d (a′–d′), immunoprecipitated with appropriate anti-tag antibody and blotted with anti-c-myc antibody (a′) or with anti-Flag-M2 antibody (b′–d′). Ctr is supernatant from untransfected cells. (f) Coomassie blue staining of fractions from culture supernatant b′ purified using nickel column. Lane 1 is culture supernatant before purification, 2 is flow through, 3 is wash, and 4–6 are different eluted fractions. (g) Western blot of panel (f) using anti-Flag M2 antibody.
Figure 6
Figure 6
Expression and purification of soluble scTR using mammalian expression system. (a) The single-chain fraction variable (scFv) domain was cloned and fused to a C-terminus HA and c-myc epitope tags. (b) Secreted scTCR that was cloned and fused to a C-terminus Flag and 6-His epitope tags. (c) and (d) the secreted scTR as described in (b), with the exception that the secreted scTR was fused to the leader sequence from GM-CSF (c) or from Ig-κ light chain (d). (e) Western blot of the culture supernatants from 293H cells transiently transfected with constructs a–d (a′–d′), immunoprecipitated with appropriate anti-tag antibody and blotted with anti-c-myc antibody (a′) or with anti-Flag-M2 antibody (b′–d′). Ctr is supernatant from untransfected cells. (f) Coomassie blue staining of fractions from culture supernatant b′ purified using nickel column. Lane 1 is culture supernatant before purification, 2 is flow through, 3 is wash, and 4–6 are different eluted fractions. (g) Western blot of panel (f) using anti-Flag M2 antibody.
Figure 6
Figure 6
Expression and purification of soluble scTR using mammalian expression system. (a) The single-chain fraction variable (scFv) domain was cloned and fused to a C-terminus HA and c-myc epitope tags. (b) Secreted scTCR that was cloned and fused to a C-terminus Flag and 6-His epitope tags. (c) and (d) the secreted scTR as described in (b), with the exception that the secreted scTR was fused to the leader sequence from GM-CSF (c) or from Ig-κ light chain (d). (e) Western blot of the culture supernatants from 293H cells transiently transfected with constructs a–d (a′–d′), immunoprecipitated with appropriate anti-tag antibody and blotted with anti-c-myc antibody (a′) or with anti-Flag-M2 antibody (b′–d′). Ctr is supernatant from untransfected cells. (f) Coomassie blue staining of fractions from culture supernatant b′ purified using nickel column. Lane 1 is culture supernatant before purification, 2 is flow through, 3 is wash, and 4–6 are different eluted fractions. (g) Western blot of panel (f) using anti-Flag M2 antibody.
Figure 6
Figure 6
Expression and purification of soluble scTR using mammalian expression system. (a) The single-chain fraction variable (scFv) domain was cloned and fused to a C-terminus HA and c-myc epitope tags. (b) Secreted scTCR that was cloned and fused to a C-terminus Flag and 6-His epitope tags. (c) and (d) the secreted scTR as described in (b), with the exception that the secreted scTR was fused to the leader sequence from GM-CSF (c) or from Ig-κ light chain (d). (e) Western blot of the culture supernatants from 293H cells transiently transfected with constructs a–d (a′–d′), immunoprecipitated with appropriate anti-tag antibody and blotted with anti-c-myc antibody (a′) or with anti-Flag-M2 antibody (b′–d′). Ctr is supernatant from untransfected cells. (f) Coomassie blue staining of fractions from culture supernatant b′ purified using nickel column. Lane 1 is culture supernatant before purification, 2 is flow through, 3 is wash, and 4–6 are different eluted fractions. (g) Western blot of panel (f) using anti-Flag M2 antibody.
Figure 6
Figure 6
Expression and purification of soluble scTR using mammalian expression system. (a) The single-chain fraction variable (scFv) domain was cloned and fused to a C-terminus HA and c-myc epitope tags. (b) Secreted scTCR that was cloned and fused to a C-terminus Flag and 6-His epitope tags. (c) and (d) the secreted scTR as described in (b), with the exception that the secreted scTR was fused to the leader sequence from GM-CSF (c) or from Ig-κ light chain (d). (e) Western blot of the culture supernatants from 293H cells transiently transfected with constructs a–d (a′–d′), immunoprecipitated with appropriate anti-tag antibody and blotted with anti-c-myc antibody (a′) or with anti-Flag-M2 antibody (b′–d′). Ctr is supernatant from untransfected cells. (f) Coomassie blue staining of fractions from culture supernatant b′ purified using nickel column. Lane 1 is culture supernatant before purification, 2 is flow through, 3 is wash, and 4–6 are different eluted fractions. (g) Western blot of panel (f) using anti-Flag M2 antibody.
Figure 6
Figure 6
Expression and purification of soluble scTR using mammalian expression system. (a) The single-chain fraction variable (scFv) domain was cloned and fused to a C-terminus HA and c-myc epitope tags. (b) Secreted scTCR that was cloned and fused to a C-terminus Flag and 6-His epitope tags. (c) and (d) the secreted scTR as described in (b), with the exception that the secreted scTR was fused to the leader sequence from GM-CSF (c) or from Ig-κ light chain (d). (e) Western blot of the culture supernatants from 293H cells transiently transfected with constructs a–d (a′–d′), immunoprecipitated with appropriate anti-tag antibody and blotted with anti-c-myc antibody (a′) or with anti-Flag-M2 antibody (b′–d′). Ctr is supernatant from untransfected cells. (f) Coomassie blue staining of fractions from culture supernatant b′ purified using nickel column. Lane 1 is culture supernatant before purification, 2 is flow through, 3 is wash, and 4–6 are different eluted fractions. (g) Western blot of panel (f) using anti-Flag M2 antibody.
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