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The Journal of Immunology, 2001, 166: 182-187.
Copyright © 2001 by The American Association of Immunologists

Redirecting Mouse CTL Against Colon Carcinoma: Superior Signaling Efficacy of Single-Chain Variable Domain Chimeras Containing TCR-{zeta} vs Fc{epsilon}RI-{gamma}1

Nicole M. Haynes*, Marie B. Snook*, Joseph A. Trapani*, Loretta Cerruti{dagger}, Stephen M. Jane{dagger}, Mark J. Smyth2,* and Phillip K. Darcy2,3,*

* Cancer Immunology, Peter MacCallum Cancer Institute, Victoria, Australia; and {dagger} Rotary Bone Marrow Research Laboratory, Royal Melbourne Hospital, Parkville, Victoria, Australia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The structurally related TCR-{zeta} and Fc receptor for IgE (Fc{epsilon}RI)-{gamma} are critical signaling components of the TCR and Fc{epsilon}RI, respectively. Although chimeric Ab receptors containing {zeta} and {gamma} signaling chains have been used to redirect CTL to tumors, a direct comparison of their relative efficacy has not previously been undertaken. Here, in naive T lymphocytes, we compare the signaling capacities of the {zeta} and {gamma} subunits within single-chain variable domain (scFv) chimeric receptors recognizing the carcinoembryonic Ag (CEA). Using a very efficient retroviral gene delivery system, high and equivalent levels of scFv-{zeta} and scFv-{gamma} receptors were expressed in T cells. Despite similar levels of expression and Ag-specific binding to colon carcinoma target cells, ligation of scFv-anti-CEA-{zeta} chimeric receptors on T cells resulted in greater cytokine production and direct cytotoxicity than activation via scFv-anti-CEA-{gamma} receptors. T cells expressing scFv-{zeta} chimeric receptors had a greater capacity to control the growth of human colon carcinoma in scid/scid mice or mouse colon adenocarcinoma in syngeneic C57BL/6 mice. Overall, these data are the first to directly compare and definitively demonstrate the enhanced potency of T cells activated via the {zeta} signaling pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Engagement of TCRs and Ig-binding FcR on T lymphocytes and myeloid/NK cells, respectively, are critical events in initiating host immune defense against virus-infected and malignant cells (1, 2). The signaling events downstream of both the TCR and FcR involves rapid phosphorylation of conserved 18-aa Ig tyrosine activation motifs (ITAM),4 situated in the cytoplasmic domains of these immune receptor complexes and the subsequent activation of Src family and ZAP-70 and Syk kinases (2, 3, 4, 5, 6, 7, 8). FcR-{gamma}- and TCR-{zeta}-chains contain one and three ITAMs, respectively, within their cytoplasmic domains; each ITAM sequence containing a pair of YXXL motifs separated by seven amino acids. It is these variable flanking amino acids that ultimately dictate, through recruitment of specific protein tyrosine kinases, the signaling capability and function of the {gamma}- and {zeta}-chains (4, 9). The subsequent induction of multiple downstream signaling cascades leads to cell activation and stimulation of critical biological effector functions such as cytolysis, cytokine release, and phagocytosis (8, 10, 11).

Chimeric receptors comprising the {gamma} and {zeta} cytoplasmic signaling chains fused to an extracellular ligand-binding domain of a heterologous receptor or single-chain Ab have served as effective tools for elucidating and comparing the structure-function relationship of these TCR-{zeta} and Fc receptor for IgE (Fc{epsilon}RI)-{gamma} subunits (11, 12, 13). In addition, these chimeras have offered excellent new possibilities for designing novel cellular immunotherapies (14, 15, 16, 17, 18, 19, 20, 21). We and others have previously shown that upon Ag ligation of these chimeric receptors, expressed in T cell lines (14, 15, 16), NK cells (17, 18), neutrophils (19), and primary T lymphocytes (20, 21), both the {gamma} and {zeta} subunits can effectively couple to signal transduction pathways. These signaling moieties are thus capable of mediating immune effector functions, equivalent to but independent of the endogenous FcR and TCR, respectively. Comparative studies of the signaling efficacy of {gamma} and {zeta} in T cell lines, the rat basophilic leukemic cell line, NK cells, and neutrophils have shown the {zeta} subunit to be more effective than {gamma} in mediating cytolysis and cytokine release in vitro (19, 22). However, to date, comparison of these structurally related signaling subunits in primary T lymphocytes has not been demonstrated, particularly in tumor models in vivo.

We have previously described a highly effective retroviral gene delivery system for efficiently expressing chimeric receptor gene constructs in mouse T lymphocytes (20). These studies demonstrated encouraging efficacy of a single-chain variable domain (scFv)-anti-carcinoembryonic Ag (CEA)-{gamma} chimera in redirecting T cell-mediated rejection of colon carcinoma (20). In this study, we have compared the efficacy of scFv-{gamma} and -{zeta} chimeric receptors, recognizing the CEA, to redirect T lymphocyte-mediated effector function both in vitro and in vivo. In keeping with our previous findings, T lymphocytes expressing the scFv-{gamma} chimeric receptor were capable of efficiently mediating rejection of colon carcinoma in an Ag-specific manner (20). Importantly however, T lymphocytes expressing the scFv-{zeta} chimeric receptors consistently demonstrated a greater capacity to mediate cytokine production, direct cytotoxicity, and tumor rejection in vivo. Thus, the TCR-{zeta} molecule was a superior signaling moiety in chimeric receptors redirecting T cell effector function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture

The human colorectal carcinoma cell lines COLO 205 and Lovo, mouse (C57BL/6 (B6)) colon adenocarcinoma MC-38 and its CEA+ transfectant, MC-38-CEA2 (Ref. 23 ; kindly provided by Dr. Jeff Schlom, National Institutes of Health, Bethesda, MD), and the B6 sarcoma cell line 24JK (kindly provided by Dr. Patrick Hwu, National Institutes of Health) were maintained in RPMI 1640 or DMEM at 37°C and 5% CO2 supplemented with the following additives: 10% (v/v) FCS, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Grand Island, NY). The retroviral packaging cell lines, GP+E86 and PA317 and the fibroblast cell line NIH3T3 were cultured in DMEM with additives. PA317 cells transduced with recombinant retroviral DNA were maintained in DMEM supplemented with 0.5 mg/ml G418 (Life Technologies). Transduced T cells were cultured in DMEM containing 100 U/ml of human rIL-2 (kindly provided by Chiron, Emeryville, CA).

Mice

Inbred B6, BALB/c, and BALB/c scid/scid (scid) mice were purchased from The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia. Mice of 4–8 wk of age were used in all experiments that were performed according to animal experimental ethics committee guidelines.

Chimeric receptor gene construction

A 767-bp fragment of DNA coding for scFv of anti-CEA and a marker epitope from c-myc was amplified by PCR from the vector MFE-23 (24) and subcloned into XbaI/BstEII-digested pRSVscFv{gamma}R (a kind gift from Zelig Eshhar, Weizmann Institute, Rehovot, Israel). The chimeric gene constructs were composed of the scFv of the anti-CEA mAb, a membrane-proximal hinge region of human CD8 and the transmembrane and cytoplasmic regions of the human Fc{epsilon}RI-{gamma} or TCR-{zeta}-chains. The scFv anti-CEA chimeric receptors were digested with SnaB1/XhoI and were subcloned into the HpaI/XhoI restriction sites of the retroviral vector, pLXSN (a kind gift from Dusty Miller, Fred Hutchinson Cancer Research Center, Seattle, WA) containing the long terminal repeat and a neomycin resistance gene under the control of an SV40 promoter.

Retroviral gene transfer of mouse spleen T lymphocytes

Stable GP+E86 ecotrophic packaging cell lines expressing either scFv-anti-CEA-{gamma} or -{zeta} receptors were isolated as described previously (20). GP+E86 clones producing ~107 cfu/ml were used for transduction of mouse spleen T lymphocytes. Spleen cells from mice were initially depleted of RBC by hypotonic lysis with NH4Cl and enriched by passing through a nylon wool syringe as described previously (25). Enriched T lymphocytes (107) were then cocultivated for 72 h with 5 x 105 viral-producing packaging cells in DMEM supplemented with 4 µg/ml polybrene, 5 µg/ml PHA (Sigma, St. Louis, MO), and 100 U/ml rIL-2. Following cocultivation, T cells were separated from adherent packaging cells, washed with DMEM and cultured in DMEM supplemented with 100 U/ml rIL-2. T cells were subsequently analyzed for transduction efficiency by flow cytometry and used for in vitro assays and in vivo experiments.

Flow cytometry

Detection of cell surface chimeric receptor expression on mouse T lymphocytes was achieved by indirect immunofluorescence with a c-myc tag Ab purified from supernatants of mouse 9E10 cells (26), followed by staining with a PE-labeled anti-mouse Ig mAb (Becton Dickinson, San Jose, CA). Cell surface phenotyping of transduced cells was determined by direct staining with Quantum Red-labeled anti-CD4 (clone H129-19; Sigma) and anti-CD8 (clone 53-6.7; Sigma) and PE-labeled anti-TCR{beta} (clone H57-597; PharMingen, San Diego, CA) mAbs as previously described (20).

Ag-specific binding, cytotoxicity, and cytokine secretion

The binding capacity of gene-modified mouse T lymphocytes was determined in a rosetting assay as described (16). The cytolytic capacity of transduced T cells was determined in a 6-h 51Cr-release assay as described (15). Mouse IFN-{gamma} secretion by scFv-modified mouse T lymphocytes after CEA ligation was detected by ELISA. Transduced T cells (106) (transduced with LXSN alone or LXSN plus scFv-anti-CEA-{gamma} or -{zeta}) were cultured with 106 Lovo (CEA+) or 24JK (CEA-) cells in 24-well plates for 20 h. Following incubation, supernatants were harvested and spun to remove cell debris. Levels of cytokine production were measured by ELISA (PharMingen) according to the suppliers specifications.

Adoptive transfer models

Three different adoptive transfer tumor models were employed. In the first model, 106 mouse 24JK sarcoma cells and/or 5 x 106 human COLO 205 colon carcinoma cells were injected s.c. into opposite flanks of groups of 5–10 scid mice. Spleen T lymphocytes (5 x 106) from BALB/c mice (transduced with LXSN vector alone or LXSN plus scFv-anti-CEA-{gamma} or -{zeta}) were injected i.v. into groups of 5–10 scid mice 6 h (day 0) and 24 h (day 1) after tumor inoculation. In the second model, 107 mouse MC-38-CEA2 colon adenocarcinoma cells were injected i.p. into groups of 5–10 B6 mice. Spleen T lymphocytes (5 x 104-105) from B6 mice (transduced with LXSN vector alone or LXSN plus scFv-anti-CEA-{gamma} or -{zeta}) were injected i.p. into groups of 5–10 syngeneic mice 6 h (day 0) and 24 h (day 1) after tumor inoculation. In the third model, 5 x 106 mouse MC-38 colon adenocarcinoma cells and/or the CEA+ transfectants, 5 x 106 MC-38-CEA2, were injected s.c. into opposite flanks of groups of 5–10 B6 mice. Spleen T lymphocytes (5 x 106) from B6 mice (transduced with LXSN vector alone or LXSN plus scFv-anti-CEA-{gamma} or -{zeta}) were injected i.v. into groups of 5–10 syngeneic mice 6 h (day 0) and 24 h (day 1), or day 3 after tumor inoculation. In the s.c. models, subsequent tumor growth was monitored daily and measured by a caliper square along the perpendicular axes of the tumors. The data were recorded as the mean tumor size (product of the two perpendicular diameters) ± SEM. In the i.p. model, mice were monitored daily for tumor ascites development, indicated by swelling of the abdomen and were culled when obvious signs of tumor growth were noted.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of chimeric scFv-anti-CEA-{gamma} and -{zeta} receptors in mouse T lymphocytes

The chimeric receptor gene constructs were composed of the scFv (VH and VL) regions of the anti-CEA mAb fused to the transmembrane and cytoplasmic regions of the human TCR-{zeta} or Fc{epsilon}RI-{gamma} signaling chains, via a CD8 hinge (Fig. 1GoA). The scFv-{gamma} and -{zeta} chimeric gene constructs were subcloned into the retroviral vector pLXSN and high titer-virus-producing GP+E86 clones were used to transduce enriched naive T lymphocytes from BALB/c or B6 mouse spleens as previously described (20). Surface expression of the scFv-anti-CEA receptors in transduced T cells was determined by flow cytometry using an anti-c-myc tag mAb directed at the tag epitope located within the extracellular domain of the receptors (Fig. 1Go, B–D). An equivalently high level of expression of the scFv-anti-CEA-{gamma} and -{zeta} chimeric receptors (ranging between 50 and 80%) was detected on T cells (mean channel fluorescence: scFv-{gamma} = 36.6 ± 5.8, scFv-{zeta} = 32.8 ± 5.4; n = 8; Fig. 1Go, B and C). Expression of either scFv chimeric receptor could not be detected on T cells transduced with the LXSN retroviral vector alone (mean channel fluorescence = 8.80 ± 2.7; n = 8; Fig. 1GoD). Consistent with previous observations (20), preferential proliferation of CD8+ spleen T cells in response to PHA/IL-2 was observed; the transduced T cell populations were consistently >80% TCR{beta}+CD8+.



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  		FIGURE 1. Expression of the chimeric scFv-anti-CEA-{gamma} and -{zeta} receptors in mouse T lymphocytes. A, Schematic representation of the scFv-anti-CEA-{gamma} and -{zeta} receptors. Each construct was composed of the VH and VL regions of the anti-CEA mAb joined by a flexible linker, a membrane-proximal hinge region of human CD8 and the transmembrane (TM) and cytoplasmic regions of the human Fc{epsilon}RI-{gamma}- and TCR-{zeta}-chains. Enriched splenic T cells from BALB/c or B6 mice were transduced by coculture with GP+E86 clones producing the recombinant retroviral vector encoding the anti-CEA-{zeta} (B), the anti-CEA-{gamma} (C) gene constructs, or the retroviral vector pLXSN alone (D). Cells were stained with the anti-tag mAb and PE-labeled sheep anti-mouse Ig (solid line) or with the PE-labeled secondary alone (dashed line), and receptor expression was analyzed by flow cytometry.

 
Effective Ag-specific binding by T cells expressing the scFv-anti-CEA-{gamma} and -{zeta} chimeric receptors

The ability of mouse T cells expressing either the scFv-anti-CEA-{gamma} or -{zeta} receptors to specifically bind CEA-expressing target cells was demonstrated in rosetting assays. Transduced BALB/c T cells (scFv-anti-CEA-{gamma} and -{zeta}) conjugated equally well with the CEA+ Lovo target cells (40 ± 3% vs 46 ± 2%; n = 4) but not with CEA- 24JK target cells (data not shown). T cells transduced with pLXSN control vector did not bind either target cell line (data not shown). These data suggested that the level of surface expression and scFv-mediated Ag binding of {gamma}- and {zeta}-containing chimeric receptors were equivalent.

Superior cytokine release and cytotoxicity mediated by scFv-{zeta}- transduced T cells

The capacity of the scFv-{gamma}- or -{zeta}-transduced T cells to secrete IFN-{gamma} was compared following specific interaction with CEA+ target cell lines (Table IGo). T cells transduced with the scFv-{zeta} chimera secreted >1.5-fold more IFN-{gamma} than T cells expressing the scFv-{gamma} receptor, following ligation of the CEA+ Lovo carcinoma cell line (Table IGo). T lymphocytes transduced with LXSN vector alone secreted <20 pg/ml IFN-{gamma} after interaction with Lovo or 24JK cells. Depletion of CD8+ T cells before interaction with Lovo target cells abolished IFN-{gamma} secretion (data not shown). Human Jurkat T leukemia cells expressing either the scFv-anti-CEA-{gamma} or -{zeta} receptors also secreted IL-2 in an Ag-specific manner. In particular, after ligation with CEA, Jurkat T cells transduced with scFv-{zeta} secreted 2-fold more IL-2 than Jurkat transduced with the scFv-{gamma} receptor (data not shown). This data demonstrated the {zeta}-containing chimera to also be more effective for cytokine production than the scFv-{gamma} chimera in a human T cell line.


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  		Table I. Optimal IFN-{gamma} secretion by scFv-{zeta}-expressing mouse T cells after CEA ligation1

 
The capacity of transduced T cells, expressing the scFv-{gamma} or -{zeta} chimeric receptors to mediate specific target cell lysis was evaluated in standard 6-h 51Cr release assays. BALB/c T cells expressing either the scFv-{gamma} or -{zeta} receptors were capable of lysing the CEA+ colon carcinoma cell line COLO 205, but not the CEA- 24JK sarcoma cell line. Interestingly, BALB/c T cells expressing the scFv-anti-CEA-{zeta} chimeric receptors were significantly more cytolytic than T cells transduced with the scFv-{gamma} chimera at all E:T ratios (Fig. 2Go, A and B). In B6 T lymphocytes (expressing equivalent levels of the scFv-{gamma} or -{zeta} receptors as detected on transduced BALB/c T cells (data not shown)), the {zeta}-containing chimera again was shown to have a greater cytolytic capacity than the {gamma}-containing chimera against the CEA+ MC-38-CEA2 mouse colon adenocarcinoma (Fig. 2GoC). No lysis of the CEA- parental MC-38 cells was evident in the presence of either effector population (Fig. 2GoD). The antigenic specificity of these responses was further demonstrated by the abrogation of T cell lysis by the addition of an anti-CEA-mAb, but not by an IgG isotype control mAb (data not shown). T cells transduced with the LXSN vector alone were unable to lyse the CEA-expressing and control target cell lines (Fig. 2Go, A–D).



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  		FIGURE 2. Superior efficacy of scFv-anti-CEA-{zeta}-transduced T cells in mediating Ag-specific lysis of CEA+ tumor cells by transduced T cells. The cytolytic function of the transduced T cells expressing the scFv-anti-CEA-{gamma} and -{zeta} receptors was evaluated in a 6-h 51Cr release assay. T cells transduced with the scFv-{gamma} ({blacksquare}) or scFv-{zeta} ({circ}) chimeric receptors lysed the CEA+ COLO 205 colon carcinoma cell line (A) but not the CEA- 24JK sarcoma cell line (B). T cells transduced with the pLXSN retrovirus alone ({square}) were unable to lyse the COLO 205 and 24JK cell lines. The lytic activity of enriched splenic T cells from B6 mice transduced with the scFv anti-CEA-{gamma} chimera ({blacksquare}), the scFv anti-CEA-{zeta} chimera ({circ}), or pLXSN alone ({square}) was evaluated against the CEA+ colon adenocarcinoma cell line MC-38-CEA2 (C) or CEA- MC-38 parental cell line (D). The spontaneous lysis was <10% in all assays. Results are expressed as specific 51Cr release ± SE (%) for triplicate samples and are representative of at least two experiments.

 
Superior efficacy of scFv-anti-CEA-{zeta}-transduced T cells in rejecting human colon carcinoma in scid mice

The signaling efficacy of the scFv-anti-CEA-{gamma} and -{zeta} chimeric receptors in stimulating T cell effector function was further evaluated in a human tumor rejection assay in vivo. Both types of transduced T cells (5 x 106) were adoptively transferred i.v. into scid mice, 6 h (day 0) and 24 h (day 1) after the subcutaneous inoculation of CEA+ COLO 205 tumor in the right flank and CEA- 24JK tumor in the left flank. As previously reported (20), the scFv-{gamma} chimeric receptor transduced effector T cells injected i.v. mediated an Ag-specific anti-tumor response against the CEA+ COLO 205 tumor xenografts, with 2 of 10 tumors completely eradicated (Fig. 3GoA). However, more striking was the effect of the scFv-{zeta}-transduced T cells, with the complete eradication of 6 of 10 COLO 205 tumors (Fig. 3GoA). Of the mice with tumors not eradicated by treatment with either population of chimera-transduced T cells, only those receiving scFv-{zeta}-transduced cells displayed a reduced tumor growth rate compared with mice receiving pLXSN-transduced T cells or no treatment. The antigenic-specificity of both scFv chimeric receptors was demonstrated by the complete lack of effect of these transduced T cells on CEA- 24JK tumors growing in the opposite flank of these mice (Fig. 3GoB). Mice with tumors eradicated by treatment were monitored for between 50 and 100 days after tumor inoculation and all remained tumor free.



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  		FIGURE 3. Optimal rejection of colon carcinoma in scid mice by redirected T cells expressing the scFv-{zeta} chimera. A, The growth of the CEA+ human COLO 205 colon carcinoma cells in groups of 5–10 scid mice injected s.c. into the right flank with 5 x 106 tumor cells. B, Mice were additionally injected in the left flank with 106 CEA- 24JK sarcoma cells ({blacksquare}). Mice were injected i.v. with 5 x 106 BALB/c T cells transduced with the pLXSN vector alone ({square}), scFv-anti-CEA-{gamma} chimera ({circ}), or scFv-anti-CEA-{zeta} chimera (•) on days 0 and 1 after tumor inoculation. Untreated mice are depicted by filled squares ({blacksquare}) for either COLO 205 (A) or 24JK (B). For all experiments, results are represented as the mean tumor size (mm2) ± SE. Arrows depict the days of T cell transfer and the number of tumors not eradicated is shown.

 
Superior efficacy of scFv-anti-CEA-{zeta}-transduced T cells in controlling syngeneic mouse adenocarcinoma

The efficacy of the scFv-anti-CEA-{gamma}- and -{zeta}-transduced T lymphocytes was evaluated following adoptive transfer into B6 mice inoculated either i.p. or s.c. with MC-38-CEA2 adenocarcinoma cells. In the first model, i.p. transfer of 5 x 105 T cells transduced with the scFv-anti-CEA-{gamma} receptor, 6 h (day 0) and 24 h (day 1) after i.p. inoculation of MC-38-CEA2 tumor, resulted in 40% survival (4 of 10 mice; Fig. 4Go). By comparison, intraperitoneal injection of T cells (5 x 105; days 0 and 1) transduced with the scFv-anti-CEA-{zeta} receptor eradicated tumors, with 70% survival (7 of 10 mice). The superior efficacy of the scFv-{zeta} chimera was further demonstrated by the 30% survival (3 of 10 mice) of mice i.p. injected with 5 x 104 (days 0 and 1) scFv-{zeta}-transduced T cells (Fig. 4Go). The i.p. injection of 5 x 104 (days 0 and 1) scFv-{gamma}-expressing T cells had little anti-tumor effect with no eradication of tumors. Both scFv-anti-CEA-{gamma}- and -{zeta}-transduced T cells had no effect on the i.p. growth of the MC-38 parental line (data not shown).



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  		FIGURE 4. Superior anti-tumor response by scFv-{zeta}-transduced T cells against i.p. syngeneic colon adenocarcinoma. The survival of B6 mice inoculated i.p. with 107 MC-38-CEA2 tumor in groups of 5–10 B6 mice. Mice were injected i.p. with 106 B6 T cells transduced with the pLXSN vector alone ({square}), 5 x 104 ({circ}) or 5 x 105 (•) scFv anti-CEA-{gamma} chimera, or 5 x 104 ({triangleup}) or 5 x 105 ({blacktriangleup}) scFv anti-CEA-{zeta} chimera on days 0 and 1 after tumor inoculation. Untreated mice are depicted by filled squares ({blacksquare}). Results are represented as the percentage of survival and arrows depict the days of T cell transfer.

 
In the second and more stringent model, the i.v. transfer of T cells transduced with scFv-anti-CEA-{gamma} effectively rejected s.c. MC-38-CEA2 tumor growth in an Ag-specific manner, consistent with our previous findings (20) (Fig. 5Go, A and B). Intravenous transfer of 5 x 106 transduced B6 scFv-{gamma} T cells 6 h (day 0) and 24 h (day 1) after tumor inoculation resulted in the complete rejection of MC-38-CEA2 tumor in 3 of 10 mice. However, T cells transduced with scFv-anti-CEA-{zeta} receptor were more effective, with 6 of 10 mice free of MC-38-CEA2 tumor (Fig. 5GoA). Again, the scFv-{zeta}-transduced T cells were also more efficient at inhibiting the growth of tumors escaping rejection. The antigenic-specificity of transduced T cells was maintained in this model with no effect on the growth of the CEA- parental MC-38 tumor in the opposite flank of these mice (Fig. 5GoB).



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  		FIGURE 5. Transduced T cells expressing the scFv-anti-CEA-{zeta} chimera are more efficient at mediating eradication of s.c. syngeneic colon adenocarcinoma. The s.c. growth of 5 x 106 (CEA+) MC-38-CEA2 (right flank) (A) and 106 (CEA-) MC-38 (left flank) (B) colon adenocarcinoma cell lines in groups of 5–10 B6 mice. Mice were injected i.v. with 5 x 106 B6 T cells transduced with the pLXSN vector alone ({blacksquare}), scFv anti-CEA-{gamma} chimera ({circ}), or scFv anti-CEA-{zeta} chimera (•) on days 0 and 1 after tumor inoculation. Untreated mice are depicted by filled squares ({blacksquare}) for either MC-38-CEA2 (A) or MC-38 (B).

 
While we have demonstrated effective early treatment of human colon carcinoma and mouse adenocarcinoma using scFv-anti-CEA-{gamma}-transduced T cells, these T cells were previously only found to be moderately effective against 3-day established tumors (20). We thus compared the efficacy of scFv-anti-CEA-{gamma}- or -{zeta}-transduced T cells against established MC-38-CEA2 tumors (Fig. 6GoA). Although no complete tumor eradications were achieved, the scFv-anti-CEA-{zeta}-transduced T cells compared favorably, with a clear improvement in the control of tumor growth (up to a 2-fold reduction in mean tumor size 5–14 days after tumor inoculation) above that observed with scFv-anti-CEA-{gamma}-transduced T cells. Growth of the CEA- parental MC-38 tumor in the opposite flank of these mice was not affected (Fig. 6GoB).



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  		FIGURE 6. Optimal growth inhibition of established s.c. syngeneic colon adenocarcinoma by scFv-anti-CEA-{zeta}-transduced T cells. The s.c. growth of 5 x 106 (CEA+) MC-38-CEA2 (right flank) (A) and 106 (CEA-) MC-38 (left flank) (B) colon adenocarcinoma cell lines in groups of 5–10 B6 mice. Mice were injected i.v. with 5 x 106 B6 T cells transduced with the pLXSN vector alone ({square}), scFv-anti-CEA-{gamma} chimera ({circ}), or scFv-anti-CEA-{zeta} chimera (•) on day 3 after tumor inoculation. Untreated mice are depicted by filled squares ({blacksquare}) for either MC-38-CEA2 (A) or MC-38 (B).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Despite existing as subunits of multicomponent surface receptors, the TCR-{zeta} and Fc{epsilon}RI-{gamma} signaling chains have the capacity to mediate critical biological effector cell activities, equivalent to but independent of the TCR and Fc{gamma}R, respectively (12, 27). Most useful has been their incorporation into chimeric immune receptors that provide effective tools for designing new cellular immunotherapies for cancer. By linking various signal transduction moieties to a constant extracellular binding domain that specifically regulates effector cell recognition of tumors, we have been able to assess the relative activity of each cytoplasmic domain in stimulating T cell function. Although both the Fc{epsilon}RI-{gamma}- and TCR-{zeta}-chains have previously been shown to be capable of autonomous activation of T cells (14, 15, 16, 17, 18, 19, 20, 21), the signaling efficacy of the {gamma} and {zeta} subunits has never been directly compared in vivo using primary T cell effectors. Importantly, in this study, high and equivalent expression of both the scFv-{zeta} and scFv-{gamma} chimeric receptors was achieved in mouse T lymphocytes using retroviral gene transduction, and we have clearly demonstrated the TCR-{zeta} molecule to be a more effective activator of T cell anti-tumor function than Fc{epsilon}RI-{gamma}. In particular, the significant improvement in the anti-tumor activity of scFv-{zeta}-transduced T cells against established tumor grafts was encouraging and efforts to further improve chimera design can be adequately tested in these tumor models. Although these tumor models suggest scFv-transduced T cells can prevent tumor initiation at a distant site, the eradication of more established tumors and their metastases is the ultimate goal of this approach.

Although primarily associated with the Fc{gamma}RI in neutrophils and macrophages (28, 29), expression of Fc{epsilon}RI-{gamma} has been detected in the cytotoxic T cell line, CTLL (30), and in T cell populations such as early thymocytes (31), NK1.1+ thymocytes and T cells (32, 33), and lymphokine-activated {gamma}{delta}-TCR+ T cells (34). These studies support a potentially broad biological role for {gamma} in T cell-mediating effector activity and thus provided scope for comparing the signaling efficacy of the {gamma} and {zeta} signaling moieties in primary T lymphocytes. The observation that the TCR-{zeta}-chain was a superior signal transducer in mouse T lymphocytes was also consistent with previous in vitro studies performed in T cell lines, neutrophils, and NK cell effector populations (19, 22). The observation that {zeta} was more effective than {gamma} may not be surprising given that the multimerization of ITAMs in the {zeta}-chain may provide a potential means of signal amplification, which may increase the sensitivity of the scFv-{zeta} chimera to ligand stimulation (35, 36, 37). A previous study has shown the qualitative differences within {gamma} and {zeta} to account for the markedly more efficient phagocytic signaling capability of the {gamma} subunit (9); however, T cell-mediated phagocytosis was not assessed in this study. An interesting future experiment will be to construct a scFv-{zeta} chimera composed of a single ITAM to determine whether ITAM multimerization normally provides the scFv-{zeta} chimera superior signaling efficacy compared with the single ITAM containing scFv-{gamma} chimera. Previously, we demonstrated the critical importance of both direct cytotoxicity mediated by perforin and T cell IFN-{gamma} production to effective tumor control in vivo (20). Importantly, in this study it was shown that IFN-{gamma} was not required for the cytotoxic capacity of scFv-transduced T cells, since those from IFN-{gamma}-deficient mice were as lytic as effectors from wild-type mice. In this light, the greater direct cytolysis and IFN-{gamma} secretion triggered by the {zeta}-chain chimera in T cells may at least in part explain the enhanced in vivo activity of these transduced T cells. It remains to be assessed whether {zeta}-containing chimeras provide a greater proliferative and more effective survival signal than scFv-{gamma} receptors. Mice cured of tumor did not resist subsequent tumor rechallenge (data not shown), suggesting that long-lived effector cells and memory cells were not present. However, repeated adoptive transfer, coadministration of T cell help/cytokines and/or modified scFv chimera design will be pursued to strive for more effective and sustained tumor protection.

Given the design of the {gamma}- and {zeta}-containing chimeras used in this study, the ability to harness the signaling machinery of the TCR may hypothetically account for the superior signaling capabilities of the {zeta} subunit in the mouse T lymphocytes. Each chimeric receptor contained the complete {gamma}- or {zeta}-chain transmembrane sequence, sufficient to enable the dimerization of the chimeras with endogenous {gamma}- or {zeta}-chains of the TCR expressed on the transduced T cells (30, 38). In our tumor model, antigenic specificity of the redirected CTL was demonstrated by the rejection of CEA+ colon carcinomas but not CEA- tumor inoculated in the opposite flank of these mice. However, we cannot discount the potential involvement of endogenous TCR signaling activity in these antitumor responses, initiated by Ag ligation of the chimeric scFv receptors. Importantly, ligation of CEA did not appear to cotrigger any endogenous TCR reactivity with self tissues in the syngeneic mouse tumor model. The higher binding affinity of scFv-receptors, compared with native TCR, for ligand could potentially influence the potency of the biochemical responses mediated by particular T cell effector populations (39, 40). We are currently assessing scFv chimera expression in TCR-transgenic T cells to determine whether endogenous T cell activity is triggered or dampened. Neither syngeneic nor xenogeneic CEA+ tumor models assessed autoimmunity via CEA ligation, since this Ag is tumor specific in B6 or scid mice, respectively. However, autoimmunity is a potential problem for this scFv approach in general, while tumor-restricted Ags remain limiting. In the future, models should be established that take this reality into consideration although bystander autoimmunity is only most accurately defined in clinical trials. Unlike vaccination strategies, if autoimmunity did arise, adoptively transferred scFv-expressing T cells could be tailored with suicide genes.

With the rapid development of new lentiviral vector systems, the transfer of scFv chimeras into nondividing and/or human T lymphocytes is now a reality. Designed chimeric immune receptors, containing cytoplasmic domains that can optimally stimulate T cell effector function, proliferation, and survival, can be tested in these settings and ultimately be exploited to enhance the potency and safety of this adoptive immunotherapy in the clinic.


   Footnotes
 
1 M.J.S. and J.A.T. are currently supported by National Health and Medical Research Council of Australia Principal Research Fellowships. S.M.J. is supported by a Wellcome Trust Senior Research Fellowship. P.K.D., M.B.S., and the work were supported by project grants from the National Health and Medical Research Council of Australia and the Anti-Cancer Council of Victoria. Back

2 M.J.S. and P.K.D. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Phillip Darcy, Cancer Immunology Research Laboratory, Peter MacCallum Cancer Institute, Locked Bag 1, A’Beckett Street, 8006, Victoria, Australia. Back

4 Abbreviations used in this paper: ITAM, Ig tyrosine activation motifs; CEA, carcinoembryonic Ag; scFv, single-chain variable domain; B6, C57BL/6. Back

Received for publication July 19, 2000. Accepted for publication October 2, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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