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Vo!. 2, 1001-1008, June 1996 Clinical Cancer Research 1001
Cytolysis of Tumor Cells Expressing the Neu/erbB-2, erbB-3, and
erbB-4 Receptors by Genetically Targeted Naive T Lymphocytes
Uwe Altenschmidt, Raif Kahl, Dirk Moritz,
Barbara S. Schnierle, Bernhard Gerstmayer,
Winfried Wels, and Bernd Groner’
Institute for Experimental Cancer Research, Tumor Biology Center,
Breisacher Strasse 177. D-79106 Freiburg, Germany
ABSTRACT
We are developing strategies to use naive T lympho-
cytes in cancer therapy. For this purpose, we are deriving T
cells with specificity of recognition for defined tumor cells.
To direct effector lymphocytes toward tumor cells, we have
manipulated the recognition specificity of naive rat and
mouse T lymphocytes and a mouse T-cell line. The cells were
stably transduced with a chimeric T-cell receptor (TCR)
component. The � chain of the TCR consists of a single
transmembrane protein with a short extracellular domain
and an intracellular domain for TCR signaling. We pro-
vided an extracellular tumor cell recognition domain to the
� chain. Human heregulin f31 (ligand to the erbB-3 and
erbB-4 receptors) and three different single-chain antibodiesspecific for the human and rat Neu/erbB-2 receptors were
used. One single-chain antibody (Cli) is directed against the
rat Neu protein, and one single-chain antibody (FRP5) is
directed against the human erbB-2 receptor. The single-
chain antibody (R-AK) directed against the Mr 14,000 fusion
protein of orthopox viruses served as a control. An efficient
procedure was devised to introduce the chimeric genes intoprimary rat and mouse T lymphocytes. Retrovirus-produc-
ing packaging cell lines were cocultured with the T cells
activated by phytohemagglutinin and interleukin 2. T-cell
lines were transduced by exposure to retrovirus-containing
supernatants from helper cell lines. Expression of the fusion
genes was determined by fluorescence-activated cell sorting
analysis More than 80% of the naive rat and mouse T cells
and 85-100% of the cells from the established T-cell lines
expressed the fusion genes within 48 h after infection. The
expression of the fusion genes was maintained for at least 10
days after infection. Target cells expressing Neu/erbB-2,
erbB-3, or erbB-4 were lysed in vitro with high specificity by
T cells expressing the corresponding recognition proteins.No selection of a marker gene is necessary to confer a
predetermined recognition specificity. The described exper-
iments are important for a gene therapy approach to cancer
treatment with autologous T cells.
INTRODUCTION
T lymphocytes are the main effector cells operative in the
recognition and elimination of syngeneic tumors ( I ). Different
tumor cells have been found to possess tumor-specific antigens,
which can be recognized by T cells (2-4). T cells are able to
penetrate the core of solid tumors and to secrete cytokines,
which activate nonspecific effector cells. The transfer of T
lymphocytes with antitumor activity into cancer patients is one
approach of adoptive immunotherapy (5-7). Tumor-infiltrating
lymphocytes, in vitro sensitized lymphocytes derived from
CTLs (8), and lymphokine-activated killer cells (9) have been
shown to possess the potential to mediate tumor regression.
In clinical trials, however, it was shown that only a fraction
of the treated patients responded to the cell transmissions (10).
The efficiency of lymphocyte-mediated tumor therapy can po-
tentially be improved by in vitro manipulation of the cells. We
are developing methods of genetic manipulation of T-cell rec-
ognition specificity to target them toward tumor cells. For this
purpose, chimeric components of the TCR2 are derived and
expressed in transduced T cells.
The TCR is a multimeric complex composed of six differ-
ent subunits, TCR-a, TCR43, CD3-y, CD3& CD3#{128},and the
CD3� chain or its alternative splice product ‘q. The TCR-a43
heterodimers recognize antigen peptides in a complex with
MHC molecules on antigen-presenting cells ( 1 1 ). Because the
TCR-a/� proteins have only marginal intracellular domains,
CD3�(1) homodimers serve as transducers to the intracellular
signaling machinery. They play a central role in the induction of
cytolysis ( I 2-15).
Recently, it was demonstrated that fusion of extracellular
ligand-binding structures with the � chain results in molecules
that can be activated by interaction with ligands ( 16-19). T
cells, which express a single-chain antibody directed against the
human erbB-2 receptor, coupled with the � chain of the TCR,
conferred cytotoxicity in vitro and in vito in a MHC-independ-
ent manner ( I 7). The target structure, the erbB-2 receptor, is
overexpressed in about 30% of human breast and ovarian car-
cinomas (20-22). erbB-2, also called Her-2, belongs to the EGF
receptor family. Members of this family comprise the EGF
receptor (EGFRIerbB- I ). erbB-2/Her-2/neu, erbB-3/Her-3, and
erbB-4/Her-4 (for reviews, see Refs. 23 and 24). In addition to
the erbB-2 protein, the erbB-3 receptor is also overexpressed in
mammary tumors and tumor cell lines (25, 26). Whereas no
specific ligand has yet been found for the erB-2 receptor, the
Received I 1/1/95: revised 1/1 7/96; accepted 3/5/96.
I To whom requests for reprints should be addressed. Phone: (49)
761-206- 1600; Fax: (49) 761-206-1699.
2 The abbreviations used are: TCR, T-cell receptor; EGF, epidermal
Fig. 1 Schematic representation of the fusion genes and the retroviral gene transfer vector pLXSN. scFv, fusion of cDNAs encoding the VH and VLto corresponding mAbs. VH and VL are joined by a short polypeptide linker sequence; L, leader sequence derived from an immunoglobulin
heavy-chain leader; Hinge, region of the CD8cx gene (29). CD3� is a signal-transducing component of the TCR-CD3 complex. Fusion gene productscould be detected by mAbs directed against the myc-tag epitope. A, scFv (Cl I) directed against the extracellular domain of the rat Neu protein. B,scFv (FRP5) directed against the extracellular domain of the human erbB-2 receptor. C, scFv (R-AK) directed against the Mr 14,000 surface proteinof the orthopox virus. D, human heregulin isoform [31 , the natural ligand to erbB-3 and erbB-4 receptors. The fusion genes are transcriptionallyregulated by the Moloney rnurine leukemia virus 5’ long terminal repeat (LTR). The vector also encodes a G418 resistance gene (neo), which isregulated by the SV4O promoter. Arrows, transcriptional start sites.
Fig. 2 Experimental strategyfor the transduction of T cells.Retroviral vectors harboring
the fusion gene inserts were in-
troduced in the packaging cell ________________line I1E by CaPO4 precipita-
tion. Enriched splenic primaryT lymphocytes from BDLX rats
or BALB/c mice were activatedwith PHA and IL-2 and trans-
duced by coculture with hightiter-producing QE clones. The _______T-cell line C196 was transducedby incubation with IL-2-sup-
plemented viral supernatants ofliE. The extent of transductionwas determined by FACS anal-
Fig. 4 Comparison of two different transduction strategies of naive rat T lymphocytes. T cells were treated with retroviral supernatant (B) or
cocultured with lIE clones producing the scFv C11:rnvc:hinge:� fusion gene (C). Untreated T cells served as controls (A). Cells were stained withmAb Mycl-9E10 directed against the myc-tag epitope of the fusion genes and with goat antimouse IgG (FITC labeled) and analyzed by FACScananalysis.
.0
EC
0)()
0)
�0
0)
Fluorescence intensity
Clinical Cancer Research 1005
I-0)E
0)U
0)
0)
scFv and heregulin fusion genes (Fig. 5). About 80% of the T
cells transduced with the fusion genes expressed the gene prod-
ucts on their cell surfaces (Fig. 5, B-E). The mean fluorescence
increased from about 6 obtained with cells transduced with
pLXSN (Fig. SA) to about 95 obtained with cells transduced
with the fusion genes.
The mouse CTL line Cl96 was infected by viral superna-
tants and selected by G4 I 8 treatment for 48 h (Fig. 1 ). There-
after, the expression of the fusion gene products on the cell
surface was determined by FACS analysis (Fig. 6). Transduc-
tion with the retroviral vector did not increase expression of the
myc-tag-containing proteins (Fig. 6A). As shown in Fig. 6, B-E,
infection of Cl96 cells with the recombinant retroviruses gen-
erated T-cell populations that expressed the fusion proteins to
nearly 100%. The mean fluorescence increased from about 3 for
cells transduced with pLXSN to about 30 for cells transduced
with recombinant vectors directing the expression of � fusion
proteins. Similar results were obtained when human Jurkat cells
were transduced by incubation with retroviruses containing su-
pernatants from PA317 cells (data not shown).
The expression of the scFv (Cli) fusion gene on primary
rat T cells was determined at different times after retroviral
infection. As shown in Fig. 7, the level of fusion gene expres-
sion did not change until day 10 (Fig. 7, D-F). Rat T cells
transduced with the retrovirus pLXSN served as a negative
control (Fig. 7, A-C).
Specific Cytotoxicity of Transduced Lymphocytes.
The cytotoxic activities of the transduced primary T lympho-
cytes from BDIX rats and the mouse T cell line Cl96 were
determined in vitro (Fig. 8). The Neu protein-expressing cancer
cell line NV2Cd derived from BDIX rats and the erbB-2-,
erbB-3-, and erbB-4-expressing mouse epithelial cell line
HCI 1RI I 1 were used as target cells. The release of 5tCr from
these cells was used as a measure of cell lysis. Syngeneic
primary T cells of BDIX origin transduced with the scFv (CII)
fusion gene lysed NV2Cd at E:T ratios of 4-100 (Fig. 8A). No
cell lysis could be measured when T cells expressing the scFV
(R-AK) fusion gene or the vector pLXSN were used. Similar
results were obtained with transduced Cl96 cells (Fig. 8C).
Fig. 5 FACS analysis of the expression of fusion genes on the cell
surface of transduced prinlary mouse T lymphocytes. T cells were
transduced by coculture with f�E clones producing the retroviral vector
pLXSN (A) or recombinant retroviral vectors harboring fusion genes
including scFv Cl I (B), scFv FRP5 (C), scFv R-AK (D), and heregulin
131 (E). Cells were stained with the mAb Mycl-9E10 directed against themyc-tag epitope of the fusion genes and with goat antimouse lgG (FITC
labeled) and analyzed by FACScan analysis (dark areas). Light areas,
cells stained only with the FITC-labeled antibody.
Naive rat lymphocytes transduced with scFv (FRP5) or heregu-
lin �3l fusion genes lysed HCI lRl 1 1 cells at E:T ratios of
0.8-100 with equal efficiencies (Fig. 8B).
Cytotoxic effects of Cl96 cells transduced with scFv
(FRP5) were determined with HCI IRI I 1 target cells at E:T
ratios of 100-20. For comparison, C196 cells transduced with
the heregulin �3l fusion gene lysed HC1 IR1 1 1 target cells at
E:T ratios of 5-100 (Fig. 8D).
The specific cytotoxic activity of the transduced primary T
cells and Cl96 cells shows that the strategies used here are able
to direct the cytotoxic activities toward tumor cells. The effi-
cient transduction of primary T cells and the grafting of target
Fig. 6 Transduction of Cl96 and analysis of the expression of fusiongenes on the cell surface by FACScan analysis. C196 T cells wereincubated with supernatants of tiE clones producing the retroviralvector pLXSN (A) or recombinant retroviral vectors harboring fusiongenes with scFv Cli (B), scFv FRP5 (C), scFv R-AK (D), and heregulin�3l (E). Cells were stained with the mAb Mycl-9E10 directed against themyc-tag epitope of the fusion genes and with goat antimouse IgG (FITC
labeled) and analyzed by FACScan analysis (dark areas). Light areas,
cells stained only with the FITC-labeled antibody.
cell recognition specificity are important prerequisites for the
clinical use of CTLs in cancer treatment.
DISCUSSION
For the successful elimination of target cells, naturally
occurring T cells require the recognition of antigen peptides
presented in MHC complexes by the TCR and the interaction of
costimulatory molecules. The ability to alter the T-cell recog-
nition specificity using chimeric receptor genes may broaden the
prospects of adoptive cellular immunotherapy to include the
treatment of viral diseases and cancer. Several reports based on
this strategy have been published (16-19).
We are expanding on these observations and demonstrate
that it is also possible to manipulate primary T cells and to
transduce them with chimeric receptor genes. When these mol-
ecules are expressed on the surface of the T cells, they provide
them with the ability to lyse specific tumor cells in a MHC-
unrestricted manner. We used the natural ligand to the erbB-3
and erbB-4 receptors, heregulin �3l, and scFvs directed against
the rat Neu protein, against the erbB-2 receptor, and against a
viral surface protein as target structures to direct T-cell recog-
nition. These molecules were fused to the signal-transducing �
chain of the TCR complex; a CD8-a hinge region was intro-
duced to improve the ligand-binding property; and a myc
tag was introduced to follow the expression of the chimeric
molecules.
The fusion genes were transduced into T cells by retrovi-
ruses. The highest efficiency (80-100%) was obtained when
naive rat or mouse T cells were cocultured with retrovirus-
producing packaging cell lines in the presence of PHA and IL-2
as activators. The transduction efficiencies we obtained were
substantially higher than those described earlier. The transduc-
tion rate of CD4� and CD8� cells derived from peripheral
su
A01
100.
10� 1#{244}2
E
i03 1(
Fluorescence intensity
Fig. 7 FACS analysis of the expression of fusion genes on the cellsurface of transduced prirnary rat T lymphocytes at different times afterviral infection. T cells were transduced for 2 days by coculture with f1Eclones producing the retroviral vector pLXSN (A-C) or recombinantretroviral vectors harboring a fusion gene including scFv Cl 1 (D-F) andcultured without flE for 8 days. Cells were stained 2 days (A and D), 5days (B and E), and 10 days (C and F) after infection with the mAbMycl-9E10 directed against the myc-tag epitope of the fusion gene andwith goat antimouse IgG (FITC labeled) and analyzed by FACScan
analysis (dark areas). Light areas, cells stained only with the FITC-
labeled antibody.
blood lymphocytes was reported to be 6-8% (45, 46). Mavilio
et a!. (47) showed that after coculture with virus-producing
helper cells, 15% of human peripheral blood lymphocytes could
be transduced. Comparable transduction efficiencies were re-
cently reported for human thymic T-cell progenitor cells (65-
85%; Ref. 42). The transductions for the T-cell lines C196 and
Jurkat were performed by incubation with retroviral superna-
tants for 24 h. The efficiencies obtained (>80%) are comparable
with the ones reported by Staal et a!. (48).
We compared the transduction efficiency of naive T cells
after coculture and treatment with retroviral supernatants. The
transduction levels reached by coculture were 2-3-fold higher
when compared with the levels reached by transduction with
viral supematants. These differences were also observed with
Jurkat cells (48). The enhanced transduction efficiency obtained
Fig. 8 Specific MHC-unrestricted lysis of tumor cell lines by trans-
duced naive rat T cells (A and B) or T-cell line C196 (C and D). Primary
T lymphocytes were transduced with the retroviral fusion genes by
coculture with the retrovirus-producing packaging cell line lIE and thecell line C196 by incubation with retrovirus-containing supernatants. Rat
T cells or C196 cells were transduced with the scFv Cl I fusion gene(antirat ,ieu; #{149}),the scFv FRP5 fusion gene (antihuman erbB-2; A). thescFv R-AK fusion gene (antipox virus, control; A), the heregulin �3l
fusion gene (#{149}),or the retroviral vector pLXSN (0). Transduced T cellswere incubated with the 5tCr-labeled rat tumor cell line NV2Cd (A andB) or the 51Cr-labeled mouse erbB-2-, erbB-3-, and erbB-4-expressingtumor cell line HC1 IR1 1 1 (C and D) as targets in 6-h cytotoxicity
assays.
by cocultivation is most likely due to an optimal infection of
viruses as a result of cell-cell contact and to the high local virus
concentrations present between the cells. Furthermore, cell-cell
contacts could enhance the early events of T-cell activation by
costimulatory molecules, which are beneficial for retroviral
DNA integration.
The transduced genes include a myc-tag recognition
epitope, which allowed the direct detection of the fusion gene
products on the surface of infected cells. Because the myc-tag
epitope is part of the constant moiety of all fusion genes we
used, it is possible to analyze cells independently from their
recognition domains. The detection of recombinant scFv fusion
proteins was also shown with mAbs specifically directed against
the scFv ( 15, 1 7, 49). It is also possible to detect and to select
transduced cells by treatment with G418, but G4l8 might have
negative influences on the biological activity of naive T cells.
Also, the limited life span of naive T cells (50) makes G4l8
selection unfavorable. To avoid these disadvantages, we applied
the transduced T cells directly to in vitro cytotoxicity assays.
The results shown in Fig. 8 show that activated, transduced
naive T lymphocytes were able to lyse tumor cells expressing
specific target structures. Transduced T cells properly process
the � fusion molecules, and cytolysis is induced on engagement
of the extracellular tumor cell recognition domains. These re-
sults complement recent studies in which chimeras of the ‘y or #{128}
chains have been joined to extracellular CD4, CD8, IL-2 recep-
tor, and CD 16 domains and expressed in transduced T lympho-
cytes and basophiles (18, 51, 52).
In this report, an efficient procedure is described to gener-
ate genetically manipulated naive T cells with a grafted recog-
nition specificity. Our results show that the cytolytic effector
machinery can be redirected toward cells expressing the Neu/
erbB-2, erbB-3, and erbB-4 receptors. These receptors are im-
portant in breast, ovarian, gastric, and colon cancer. The effi-
cient provision of tumor cell-specific recognition specificity to
primary T cells moves the strategy closer to become a useful
therapeutic tool for cancer treatment.
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