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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
growth factor; mAb, monoclonal antibody; scFv, single-chain antibody;
IL-2. interleukin 2; FACS, fluorescence-activated cell sorting; PHA,
phytohemagglutinin; VH, heavy-chain variable domain; V1. light-chain
variable domain; myc-tag, human c-invc epitope.
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1002 T-Cell-mediated Tumor Cell Lysis
erbB-3 and erbB-4 receptors are specifically activated by the
heregulins (27, 28).
The principle of MHC-independent tumor cell lysis with
genetically manipulated CTLs has been established by several
laboratories (16-19). We are concerned with the requirements
for the practical application of this strategy to cancer treatment.
To improve and expand this strategy, we describe the use of
primary T cells, and we present the construction, transduction,
and expression of four � chain fusion molecules. These mole-
cules were transduced into naive T cells and conferred the
MHC-unrestricted tumor cell lysis in vitro. We used as recog-
nition domains either the natural ligand to erbB-3 and erbB-4,
human heregulin [3 1 , or scFvs specific for the rat Neu protein,
specific for human erbB-2, or specific for a viral surface protein.
These binding structures were linked to a myc-tag recognition
epitope and the � chain of TCR-CD3 complexes. The immuno-
globulin-like hinge region of the CD8a domain (29) was in-
serted as a flexible spacer. � fusion genes were transduced in
naive rat and mouse T cells and a mouse CTL line. We inves-
tigated the conditions that yielded the highest transduction ef-
ficiency of the naive cells. A procedure was established with
which 85-100% of target cells could be transduced. Transduced
T cells expressed the fusion gene products, and in vitro, these
cells efficiently lysed syngeneic rat tumor cells expressing the
Neu protein or mouse fibroblasts expressing erbB-2, erbB-3,
and erbB-4. Our studies show that the majority of naive T
lymphocytes can be genetically manipulated without further
selection to gain new target cell recognition specificity.
MATERIALS AND METHODS
Cell Lines and Culture Conditions. Cl96 is a C57BL/6
mouse (H-2�’)-derived CTL line with H-2K�’-restricted specific-
ity for P815 (H-2”) mastocytoma cells (30). C196 cells were
maintained in DMEM (Life Technologies, Inc., Eggenstein,
Germany) with 10% FCS (Life Technologies, Eggenstein, Ger-
many) and 3% cell-free supernatant of murine IL-2-secreting
X63Ag8-653 cells (31). The retroviral packaging cell lines ��E
(32) and PA317 (33), the fibroblast cell line NIH3T3, and the
BDIX rat neuronoma cell line NV2Cd3 were cultured in DMEM
with 10% FCS. HC1 lRl 1 1 cells, transfectants of the HC1 1 cell
line expressing the human erbB-2 receptor, were cultured in
DMEM supplemented with 0.4 mg/mI neomycin analogue 0418
sulfate (geniticin; Life Technologies, Gaithersburg, MD; Ref.
34). flE or PA317 cells, transfected with recombinant retroviral
DNA, were cultured in DMEM with 0.6 mg/ml G418. Enriched
T cells were cultivated in DMEM with 10% FCS, 50 p.M
�3-mercaptoethanol (Life Technologies, Eggenstein, Germany),
10 m�t HEPES buffer (Life Technologies, Eggenstein, Germa-
ny), and 60 IU/ml human IL-2 (PBH, Hanover, Germany).
Animals. Male BDIX rats (about 200 g) and 6-8-week
old BALB/c female mice (H�2b) were obtained from Charles
River Breeding Laboratories (Sulzfeld, Germany).
3 A. Y. Nikitin, J. J. Jin, J. Papewalis, S. M. Prkopenko, A. Pozharisski,
and M. F. Rajewsky. Wild-type neu transgene counteracts mutant ho-mologue in malignant transformation of rat Schwann cells, submittedfor publication.
Construction of � Chain Fusion Genes. The retroviral
vector pLXSN has been described (33). The vector is named
based on the order of genetic elements that it encodes: L, long
terminal repeat promoter; X, cloning site for DNA insertion; 5,
5V40 promoter; and N, neomycin phosphotransferase gene
(neo). The Cl 1 antirat Neu antibody (35), the FRPS antihuman
erbB-2 antibody (36, 37), and the R-AK anti orthopox virus
antibody (a gift from C-P. Czerny, University of Munich,
Munich, Germany; Ref. 38) have been described. VH and VL
were derived from PCR amplifications using oligonucleotide
primers corresponding to the 5’ and 3’ consensus sequences of
the immunoglobulin V region and joined by a flexible linker, as
described (37). All scFvs were subcloned into pWWI52 as a
PstI-XbaI fragment (29). A fragment of the heregulin isoform
131 (70 amino acids) was cloned as described recently (39, 40).
The hinge/c fusion gene (41) was complemented by myc-tag,
which is recognized by the mAb Mycl-9E10 (42). All fusion
genes (Fig. I) were ligated as SalI-SrnaI fragments into the
retroviral vector pLXSN.
Enrichment of T Cells from Spleen Tissue. Spleencells from rats and mice were depleted of RBC by hypotonic
lysis with NH4CI and subsequently passed through a nylon-wool
syringe (43). The enriched cell preparation was analyzed by
FACS analysis and contained more than 85% T cells (TCR�),
less than 5% B cells (immunoglobulin�), and about 10% other
cells.
Retroviral Gene Transfer. Recombinant proviral DNA
was transfected into the fIE packaging cell line (32) by CaPO4
precipitation. The PA317 amphotropic packaging cell lines (33)
were infected by incubation with retroviral particles present in
the supernatants of transfected I1E cell cultures for 24 h in the
presence of 8 p.g/ml polybrene (Sigma Chemical Co., Deisen-
hofen, Germany). Virus titers produced by tzeo-resistant fIE and
PA3 17 cell clones were determined. NIH3T3 cells were infected
and neomycin-resistant cell clones were quantitated after addi-
tion of the neomycin analogue 041 8 at 1 mg/mI to the culture
medium. C196 cells (106) were infected with viral supernatants
of transfected f�E clones. They were grown for 24 h in medium
with IL-2 and polybrene. The virus-producing f�E cells had
been cultured for 24 h without G418 (Fig. 2). All supernatants
were filtered through a 0.22-p.m filter (Millipore, Eschborn,
Germany).
High-titer, neo-resistant CIE clones producing about I 0�
viral particles/ml in 24 h were selected and used for the cocul-
tivation experiments. Enriched primary T lymphocytes (106)
and l0� lIE cells (semiconfluent) were cocultured for 48 h in
complete medium (Fig. 2). Before the cocultivation, liE clones
were grown for 24 h in medium without neomycin. T cells were
separated from adherent I1E cells by careful pipetting and
grown in suspension with 5 p.g/ml PHA (Sigma), 60 IU/ml
recombinant human IL-2, and 8 p.g/ml polybrene. They were
used for the FACS analysis and cytotoxicity assays.
Antibodies and FACS Analysis. The following mAbs
were used for FACS analysis: mouse antirat CD4 (phyco-
erythrin labeled); mouse antirat CD8 (FITC labeled); mouse
antirat IgG (FITC labeled); goat antimouse TCR (FITC labeled);
and goat antimouse IgG (FITC labeled). All mAbs were pur-
chased from PharMingen (San Diego, CA). The mAb R73
specific for the rat TCR was a gift from T Hunig (University of
Research. on April 14, 2020. © 1996 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
scFv(C1 1 ):myc:hinge:#{231}
B
jH�r�:eoHLTR�sv40 promoter
c Hs�� H LTR
4 scFv(R-AK):myc:hinge:#{231} -�
HRG:myc:hinge:#{231}
.. ., , ..r-�
retroviral DNA (+lnsert) animals
� surgery
cell lines (C196, Jurkat)
c:x:IXD
retroviralsupernatant
IL-2 � i�:i -
oootransduced cell lines
CaPO4
transfection
packaging celiline ciE
spleen
� homogenization
0000enriched T cells
activation:co-culture + PHA
+ IL-2
�a- transduced, activatedI lymphocytes
Clinical Cancer Research 1003
FACS-analysis (a myc)in vitro cytotoxicity
A
� LTR� VH I� VL � Hinge
Leader Linker myc-tag
�
� LTR HilVH �VL M�Hinge�
C
� LTR���L� VH � VL M� Hinge� � HSP� HLTR
D
� LTR��L�Here�uIin �ll M� Hinge � � H� neo HLTR
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-
ysis. Cytotoxicity assays werecarried out.
Research. on April 14, 2020. © 1996 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
I
1004 T-Cell-mediated Tumor Cell Lysis
Table I Determination of retroviral titers with NIH3T3 fibroblasts
Supernatants of flE PA317 clones were applied to NIH3T3 cells.G41 8-resistant NIH3T3 clones were selected and counted.
Retroviral constructs
Viral tite r (cfu/ml)
liE PA3 17
pLXSN 8 x i0� 3 x l0�
pLXSN + scFv(Cl l):rnyc:hinge:�pLXSN + scFv(FRP5):myc;hinge:�
7 x l0’�9 X l0�
3 X l0�2 x i0�
pLXSN + scFv(R-AT):myc:hinge:�
pLXSN + HGR:rnyc:hinge:�l0�
2 x l0�
4 X l0�
l0�
I �IA��Tir�FT:�D�T�� 100 101 102 1o� iO� 1�- � � � 104 100 101 102 103 io�
.�
Fluorescence iIltCIlSity
WUrzburg, Wurzburg, Germany). mAb Mycl-9E10 directed
against human myc was purified from supernatants of mouse
9E10 cells as described (42).
For FACS analysis, cells (5 X l0�-l0�) were reacted for 45
mm at 4#{176}Cwith S p.g Mycl-9ElO and antimouse IgG (FITC
labeled) or directly with a fluorescent-conjugated mAb, as de-
scribed by the manufacturers. Stained cells were washed twice
in PBS supplemented with 5% FCS and analyzed for antibody
binding by flow cytometry using a FACScan (Becton Dickin-
son, Heidelberg, Germany).
Cytotoxicity Assay. Cytotoxicity of T cells against
NV2Cd and HC1 lRl I I target cells were measured by 5tCr
release assays, as described recently (44). Effector T cells were
incubated at various E:T cell ratios with 7.5 X l0� 5tCr-labeled
target cells for 6 h at 37#{176}C.Thereafter, 100 p.1 cell-free super-
natants were removed and counted in a gamma counter. Specific
release was calculated using the formula:
% specific lysis =
(experimental release - spontaneous release). X100%
(maximal release - spontaneous release)
The maximal release is the release in 1 N HC1, and the sponta-
neous release is the release in the presence of medium only.
RESULTS
Construction of � Chain Fusion Genes. Three fusion
genes consisting of a tumor cell recognition function and the �
chain of the TCR complex were constructed (Fig. 1). The tumor
cell recognition functions are provided by the scFv Cl 1 directed
against the rat Neu protein and the scFv FRP5 directed against
human erbB-2. ScFv R-AK is directed against the Mr 14,000
surface protein of the orthopox virus and served as a control. All
scFvs are composed of the VH and VL of the mAb joined by a
1 5-amino acid linker sequence. One construct was generated in
which the sequence of human heregulin �1 (40), the natural
ligand of erbB-3 and erbB-4 receptors, served as a tumor cell
recognition domain. A myc-tag recognition epitope was in-
cluded into the constructs to facilitate the detection of their
expression with a myc-specific mAb. The myc-tag epitope is
recognized by the mAb 9ElO (42). This antibody was used to
determine the expression of the fusion proteins on the surface of
transduced T cells. A spacer domain, the hinge region, was
included in the constructs, because it was previously shown that
it enhances ligand recognition (29).
Fig. 3 Expression of � fusion genes on the cell surface of transduced
primary rat T lymphocytes. T cells were transduced by coculture with
lIE clones, producing the retroviral vector pLXSN (A) or recombinant
retroviral vectors harboring the fusion genes with scFv C 1 1 (B), scFv
FRP5 (C), scFv R-AK (D), and heregulin �3l (F). Cells were stained
with the mAb Mycl-9E10 directed against the myc-tag epitope of thefusion 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.
Retroviral Transduction of T Cells and Analysis of theExpression of the � Fusion Genes. The retroviral vector
pLXSN was used for gene transfer into T cells (Fig. 2). Enriched
naive lymphocytes from BDIX rats or BALB/c mice and the
established murine CTL line C196 were transduced with recom-
binant retroviruses. High-titer, virus-producing clones of the
packaging cell line fIE were selected by G4l8 treatment, and
the viral titers were determined (Table I ). Primary rat and
mouse T lymphocytes were transduced with recombinant or
parental retroviruses in the presence of the activating agents
PHA and IL-2 and coculture with retrovirus-producing I1E cells
(Fig. 2). After careful separation of the T cells from the adherent
liE cells, the expression of the fusion genes was determined by
FACS analysis. No further G418 selection was applied.
The mAb Mycl-9ElO was used to detect the expression of
the recombinant fusion proteins on the cell surface of the rat
lymphocytes (Fig. 3, B-E). Cells transduced with the retroviral
vector pLXSN do not express the epitope recognized by 9E10
and served as a negative control (Fig. 3A). The majority of the
activated T cells (>80%) expressed the gene constructs. The
mean fluorescence increased from about 18 obtained with con-
trol cells transduced with pLXSN to about 90-150 for the cells
transduced with � fusion genes. The transduced T-cell popula-
tions consisted of 60% ± 5% CD4� and 40% ± 5% CD8� T
cells (data not shown). Two different transduction strategies for
the scFv (Cli) fusion gene were compared: infection of T cells
with retroviral supernatants; and cocultivation with virus-pro-
ducing helper cells (Fig. 4). The transduction efficiency of naive
rat T lymphocytes, which were transduced by coculture with
virus-producing 1�1E cells (Fig. 4C), was 2-3-fold higher when
compared with T cells, which were treated with the retrovirus-
containing supernatants (Fig. 4B).
Primary mouse T lymphocytes were transduced with the
vector pLXSN or the retrovirus directing the expression of the
Research. on April 14, 2020. © 1996 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
uninfected supernatant CO- culture
Fluorescence intensity
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
Research. on April 14, 2020. © 1996 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
pLXSN scFv (Cli)
0A
0.t � _� � -.-- 0� �
100 10� 102 i03 i04 100 1...
I ‘aI
102 10�i0�
0)
--0)U0)
0)
1k:100 101 102 103 1C
50
0
IA
(4
C
I�-��-‘---- od��
) 101 102 103 10’� 1� 1
F
J2 io� io�
1006 T-Cell-mediated Tumor Cell Lysis
�C�A(�J bk..:.] �::i� 100 101 102 io3 10” 100 10� 102 1o� i0� 100 101 iOZ iO) 104�80L��1 ____
100 101 102 io’ i0� i�#{176} 101 10� i03 10�
Fluorescence intensity
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
Research. on April 14, 2020. © 1996 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
NV2Cd HC11R1#11
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C.)C)
C)I.)C.
60
50
40
30
20
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0
60
50
40
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Clinical Cancer Research 1007
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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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1996;2:1001-1008. Clin Cancer Res U Altenschmidt, R Kahl, D Moritz, et al. erbB-4 receptors by genetically targeted naive T lymphocytes.Cytolysis of tumor cells expressing the Neu/erbB-2, erbB-3, and
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