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Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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ß TCR from a MART-1-specific,
HLA-A2-restricted, human T cell clone have been efficiently transferred
and expressed in human PBL. These retrovirally transduced PBL cultures
were MART-1 peptide reactive, and most cultures recognized
HLA-A2+ melanoma lines. Limiting dilution clones were
generated from three bulk transduced PBL cultures to investigate the
function of individual clones within the transduced cultures.
Twenty-nine of 29 CD8+ clones specifically secreted IFN-
in response to T2 cells pulsed with MART-1(27–35) peptide,
and 23 of 29 specifically secreted IFN- in response to
HLA-A2+ melanoma lines. Additionally, 23 of 29
CD8+ clones lysed T2 cells pulsed with the
MART-1(27–35) peptide and 15 of 29 lysed the
HLA-A2+ melanoma line 888. CD4+ clones
specifically secreted IFN- in response to T2 cells pulsed with the
MART-1(27–35) peptide. TCR gene transfer to patient PBL
can produce CTL with anti-tumor reactivity in vitro and could
potentially offer a treatment for patients with metastatic melanoma.
This approach could also be applied to the treatment of other tumors
and viral infections. Additionally, TCR gene transfer offers unique
opportunities to study the fate of adoptively transferred T cells in
vivo. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Adoptive transfer of TIL requires that T cells be isolated and expanded from individual patients. However, not all patients have accessible tumor lesions of sufficient size (>3 cm) to provide an adequate number of T cells for expansion. In addition, tumor-specific TIL can only be obtained from 50% of TIL cultures. Consequently, TIL therapy is a viable treatment option for only 35% of melanoma patients. An alternative approach would be to stimulate and expand PBL in vitro with allogeneic human melanoma lines. These mixed lymphocyte tumor cell cultures can generate tumor-reactive CTL, but therapeutic cell numbers are difficult to attain (12, 13). Another approach is to vaccinate patients with a TAA-peptide and isolate TAA reactive CTL from PBMC by in vitro stimulation with TAA peptides (M. Dudley, personal communication). All these approaches require TIL or CTL clones to be generated and expanded for every patient and therefore may be impractical for treating a large number of patients.
We are developing alternative strategies to target TAAs by genetically
modifying a patients’ PBLs to produce anti-tumor reactivity. Our
intention is to redirect the specificity of autologous peripheral blood
T cells by the transfer of TCR genes from TAA-specific T cell clones
into patient PBLs. We have previously described the cloning of the TCR
genes from a tumor-reactive CTL clone (clone 5) derived from a TIL
culture from patient 501. The clone 5 TCR is HLA-A2 restricted and is
specific for the m9-27 peptide epitope of the melanoma-associated Ag
MART-1, which is expressed by most melanoma tumors (14, 15).
Transfection of the and ß TCR genes from clone 5 into Jurkat
cells resulted in the expression of a functional TCR on the cell
surface (16). These experiments demonstrated that TCR gene transfer can
result in the reconstitution of a functional TCR in transfected
cells.
Retroviral vectors have been successfully used to transduce T cells
(17, 18). A retrovirus that encodes the clone 5 TCR - and ß-chains
has now been constructed, and human PBL transduced with the clone 5 TCR
genes recognized melanoma cells in vitro. To examine the reactivity of
these gene-modified T cells, T cell clones were generated from
transduced PBL cultures. CD4+ and
CD8+ T cell clones were isolated that
specifically secreted cytokine in response to tumor targets and/or T2
cells pulsed with m9-27 peptide. In addition, 23 of 29
CD8+ clones lysed T2 cells pulsed with the m9-27
peptide, and 15 of 29 CD8+ clones lysed an
HLA-A2+ melanoma line. These results demonstrate
that TCR gene transfer to patient PBL is feasible and can result in CTL
with anti-tumor reactivity in vitro. This strategy offers a
potential therapy for metastatic melanoma.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A modified SAMEN' backbone was used (Fig. 1
) (19). The full-length clone 5
-chain cDNA was derived from pTA-V1 plasmid as a
XhoI/SalI fragment and ligated into the
polylinker of the SAMEN' vector producing the V1-SAMEN' construct.
The SR promoter and full-length clone 5 ß-chain cDNA was isolated
from the pcDL-Vß7 plasmid and inserted into a
SalI/BglII site engineered downstream of the
neomycin resistance gene in the V1-SAMEN' construct producing the A7
virus construct. The PG13 retrovirus producer cell line was transfected
with the A7 virus construct, and high titer clones were isolated (20).
A7/PG13 clone 6 was grown to 80% confluence in DMEM medium (Biofluids,
Rockville, MD) supplemented with 10% FBS (Life Technologies, Grand
Island, NY) and penicillin (100 U/ml)-streptomycin (100
µg/ml)-glutamine (2.92 mg/ml) (Life Technologies). Then, 18 h
before supernatant was to be harvested for PBL transductions, the media
was exchanged with fresh media.
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PBL were transduced using an adaption of the method described by Bunnell et al. (21). Briefly, human PBMC were isolated from buffy coats from normal donors at the National Institutes of Health Clinical Center by centrifugation through lymphocyte separation medium (Organon Teknika, Durham, NC). PBMC were cultured at 1 x 106 cells/ml for 72 h in AIM-V serum-free medium (Life Technologies) supplemented with 600 IU/ml IL-2 (Cetus, Emeryville, CA) and 10 ng/ml anti-CD3 mAb (OKT3; Ortho Biotech, Raritan, NJ). On day 3, cells were harvested and resuspended at 1 x 106 cells/ml in 24-well plates in retroviral supernatant containing 8 µg/ml polybrene and 600 IU/ml IL-2. Plates were centrifuged at 1000 x g at 32°C for 90 min and then incubated overnight at 37°C in a humidified, 5% CO2 incubator. This transduction procedure was repeated for a further 2 days. On day 6, the cells were harvested and resuspended at 1 x 106 cells/ml in AIM-V supplemented with 600 IU/ml IL-2. On day 8, cell concentration was adjusted to 1 x 106 cells/ml in AIM-V supplemented with 600 IU/ml IL-2 and 0.5 mg/ml geneticin (Life Technologies). PBL were selected in geneticin for 5 days, and the percent of live cells was monitored daily and their concentration was adjusted to 106 cells/ml. Following selection, on day 13, PBL were resuspended in AIM-V supplemented with 600 IU/ml IL-2. On day 15, PBL were tested in cytokine release assays. Bulk PBL cultures were cloned on day 16–18.
Limiting dilution cloning of transduced PBL cultures
Transduced PBL were plated at 10, 1, and 0.3 cells per well in 96-well microtiter plates in 0.2 ml per well. Cells were expanded using anti-CD3 stimulation following the method of Walter et al. (22). Briefly, PBL were plated in RPMI 1640 (Biofliuds) containing 11% heat-inactivated human pooled AB serum (Pel Freez, Brown Deer, WI), penicillin (100 U/ml)-streptomycin (100 µg/ml)-glutamine (2.92 mg/ml) (Life Technologies), 25 mM HEPES, 25 µM 2-ME, with 2.5 x 105 irradiated allogeneic PBMC (100 Gy) per ml, 5 x 104 irradiated allogeneic EBV-B cells (100 Gy) per ml, and 30 ng/ml anti-CD3 mAb (OKT3; Ortho Biotech). The following day, 120 IU/ml IL-2 was added. On day 5, the medium was exchanged and fresh medium containing 120 IU/ml IL-2, without OKT3, was added. On day 8, fresh IL-2 was added to yield a final concentration of 120 IU/ml. Cells were tested for reactivity on day 12 in cytokine release assays and peptide-specific/tumor-specific cloids were restimulated as described above. To expand the reactive cloids, the culture volume was increased and the numbers of PBMC and EBV-B were adjusted accordingly.
Tumor cell lines
HLA-A2-positive and -negative human melanoma cell lines expressing MART-1 and gp100 were established in the Surgery Branch from resected tumor lesions as previously described (23). 397 Mel (HLA-A2-), 397-A2 (HLA-A2- line transfected to express HLA-A2), 624–28 (HLA-A2-), 624–38 (HLA-A2+), 888 Mel (HLA-A2-), 888-A2 (HLA-A2- line transfected to express HLA-A2), 1011 Mel (HLA-A2-), 1088 Mel (HLA-A2+), and 1300 Mel (HLA-A2+) were maintained in complete medium consisting of RPMI 1640 medium supplemented with heat-inactivated 10% FBS (Biolfuids), penicillin (100 U/ml)-streptomycin (100 µg/ml)-L-glutamine (2.92 mg/ml) (Life Technologies).
Bulk TIL cultures
TIL cultures were grown as previously described (24). Briefly, tumor samples were enzymatically digested to yield a single-cell suspension, and the cells were grown in AIM-V medium (Life Technologies) supplemented with 10% heat-inactivated pooled human AB serum (Sigma, St. Louis, MO) and 6000 IU/ml IL-2 (Cetus).
Peptides
Peptides were synthesized on a model 422 peptide synthesizer (Gilson, Worthington, OH) using solid phase methods, as previously (25). The sequences of the peptides used in this study are as follows: gp100 g9-209 (ITDQVPFSV) (26); MART-1 m9-27 (AAGIGILTV) (27); tyrosinase 369-D (YMDGTMSQV) (28).
Assessment of culture reactivity by cytokine release
PBL cultures and cloids were tested for reactivity in cytokine
release assays using commercially available ELISA kits (IL-2 and
GM-CSF, R&D Systems, Minneapolis, MN; IFN-, IL-4, IL-10, and
TNF-, Endogen, Cambridge, MA). Cytokine release was measured with T2
cells either alone or pulsed with peptide (2 µg/ml, or as described
in figure legends) in complete medium for 2–3 h at 37°C. Cells were
also tested for reactivity with MART-1- and gp100-expressing melanoma
lines. For "screening" growth-positive cloids for further
expansion, cloids were not counted, a quarter of the well volume was
taken for the assays against 10,000 888 A2+ and
888 A2- melanoma cells and/or 10,000 T2 cells
pulsed with m9-27 peptide or an irrelevant peptide, in a 0.2-ml culture
volume. For other assays, 100,000 responder cells and 100,000
stimulator cells were used in a 1.0-ml culture volume. Stimulator cells
and responder cells were cocultured for 24 h. Cytokine secretion
was measured in culture supernatants.
Assessment of reactivity by lysis assay
PBL cloids were tested for their ability to lyse
HLA-A2+, MART-1+ targets in
51Cr-release assays. Target cells were T2 cells
pulsed with either the MART-1 m9-27 peptide (2 µg/ml) or an
irrelevant peptide (either gp100 g9-209 or tyrosinase 369-D), and
melanoma cell lines 888 HLA-A2- and 888
HLA-A2+, as previously described (29). Briefly,
targets were labeled for 90 min with 200 µCi
51Cr (10–35 mCi/ml, Amersham, Arlington Heights,
IL) per 1 x 107 cells, washed three times
with HBSS and then plated in triplicate with responders at the
following E:T ratios: 80:1, 20:1, 5:1, and 1.25:1, or as described in
figure legends. Plates were incubated for 4 h at 37°C and then
supernatants were harvested. 51Cr release was
measured in supernatants on a Wallac 1470 automatic counter
(Wallac, Gaithersburg, MD), and percent specific lysis was
calculated.
Cell surface immunofluorescence
Bulk PBL cultures and cloids were stained with the following Abs: FITC isotype control, PE isotype control, human CD8 FITC, and human CD4 PE (Becton Dickinson, San Jose, CA). Cell surface immunofluorescence was measured on a FACScan flow cytometer (Becton Dickinson).
Transduction efficiency measured by competitive PCR (cPCR) analysis
A genomic DNA competitor for cPCR analysis of transduction efficiency was generated from the clone 5 TCR ß-chain cDNA by PCR ( M. I. Nishimura, manuscript in preparation). The competitor is identical in sequence to the clone 5 ß-chain but contains a 50-bp deletion in the 5' end of the constant region, to allow discrimination of the competitor and clone 5 ß-chain PCR products on agarose gels. DNA was extracted from transduced PBL cultures before and after selection in G418, as previously described (30). Briefly, 1 x 106 cells were suspended in 1x PCR buffer containing 0.1 mg/ml proteinase K (Life Technologies) and 0.5% Tween 20 (J. T. Baker, Phillipsburg, NJ) in a 200-µl volume and were incubated at 56°C for 45 min followed by 70°C for 15 min. Then, 1 µl of DNA was amplified in two series of cPCR reactions in a 50-µl volume containing 1x PCR buffer (Life Technologies), 2.5 U DNA Taq polymerase (Life Technologies), 200 µM dNTP (Life Technologies), and 400 nM of both forward and reverse primers. Series 1 used a complementarity-determining region-3 (CDR-3)-specific forward primer (TIL 5b VDJ; 5'-GATCTCCTGAGTTGGGATGA-3') and a constant region specific reverse primer (Cßcon I1R, 5' CCACCTTGTCCACTCTGGC-3'). Series 2 used a constant region forward primer (CßF' 5'-GTTCCCACCCGAGGTCGC-3') and the same constant region reverse primer (Cßcon I1R). The amount of sample DNA was held constant, and the competitor was serially diluted in each series of PCR reactions. PCR was performed in a Perkin-Elmer 9600 DNA thermocycler (Perkin-Elmer, Foster City, CA) and consisted of 35 cycles of 92°C denaturation for 30 s, 60°C annealing for 30 s, and 72°C extension for 1 min. Products were electrophoresed on 2% agarose gels and visualized with ethidium bromide staining. Gels were documented using a Stratagene Eagle-eye (Stratagene, La Jolla, CA), and the amount of PCR product in template and competitor bands was measured using ImageQuaNT software (Molecular Dynamics, Sunnyvale, CA). Regression analyses were performed with Excel software (Microsoft, Redwood, WA).
TCR Vß (BV) subfamily analysis of cloids
The official nomenclature proposed by the International Union of Immunological Societies (IUIS) subcommitee on nomenclature has been used throughout this manuscript (31). Designation of TCR ß gene segments was according to Arden et al. (32). Total cellular RNA was isolated using Trizol reagent, as per manufacturers instructions (Life Technologies). cDNA was synthesized using oligo(dT) (12, 13, 14, 15, 16, 17, 18) and Superscript Preamplification System reagents (Life Technologies). Oligonucleotide sequences of the BV subfamily-specific PCR primers and PCR methodology were as previously described (M. D. McKee et al., manuscript in preparation). Briefly, 1 µl of cDNA was amplified in a 50-µl reaction volume containing 2.5 U Taq DNA polymerase (Life Technologies), 1x PCR buffer, 200 µM dNTP (Life Technologies), and 400 nM of both forward and reverse primer. Amplifications were performed with a Perkin-Elmer 9600 DNA thermocycler (Perkin-Elmer) under the following conditions: 35 cycles of 92°C denaturation for 30 s, 60°C annealing for 30 s, and 72°C extension for 1 min. PCR products were resolved on 2% agarose gels and visualized using ethidium bromide staining.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- and
ß-chain cDNAs and the neomycin resistance gene as a selectable marker
(Fig. 1
). PBL from three normal donors were stimulated with
anti-CD3 Ab and IL-2 and were transduced with the A7 virus (PBL 65
was transduced separately from PBL 66 and 67). The percentage of
transduced cells was measured before and after selection with G418 by
cPCR. Twenty-two to 38% of the unselected cells were transduced, and,
following G418 selection, 60–92% of the cells were transduced (data
not shown). Transduced and selected PBL cultures were 80–95%
CD8+ T cells by immunofluorescence analysis (data
not shown). Cytokine release assays with bulk PBL cultures demonstrated
that transduction with the clone 5 TCR genes conferred Ag-specific
reactivity to the cultures (Table I). In
two separate transduction experiments, we successfully transferred a
functional MART-1-reactive TCR to normal PBL-derived T cells as
demonstrated by their specific recognition of T2 cells pulsed with the
m9-27 peptide (Table I). In addition, the PBL-66 and PBL-67 cells
recovered from the second transduction showed specific IFN-
secretion in response to HLA-A2+ melanoma lines
expressing MART-1 (Table I ). These results demonstrate that peripheral
blood T cells from normal donors can be genetically modified to react
with a human melanoma Ag.
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in response to T2
cells pulsed with m9-27 peptide. Approximately half of the peptide
reactive clones, 15 of 34 from PBL-66 and 20 of 47 from PBL-67 (PBL 65
clones were not tested), also specifically released IFN- when
stimulated by 888-A2+ MEL. Therefore, a high
percentage of cells from each of the transduced PBL populations were Ag
reactive.
A representative group of reactive clones from each transduction were
expanded to further characterize their phenotype and Ag reactivity.
These results are summarized in Table II.
Immunofluorescence analysis revealed that all of the clones tested
from PBL-66 and PBL-67 were CD8+ (9 of 9 and 12
of 12, respectively), while clones from PBL-65 were
CD4+ (4 of 13), CD8+ (8 of
13), or a mixture of CD4+ and
CD8+ T cells (1 of 13). In cytokine release
assays, each of the 29 CD8+ clones secreted
IFN- in response to m9-27 peptide-pulsed T2 cells and 18 of 29
CD8+ clones secreted IFN- in response to tumor
cells (Table II). In 51Cr release assays, 23 of
the 29 CD8+ clones lysed T2 cells pulsed with
m9-27 peptide, and 15 of the 29 CD8+ clones lysed
the HLA-A2+ melanoma line 888 MEL (Table II).
None of the CD4+ clones lysed m9-27
peptide-pulsed T2 cells or 888-A2 melanoma cells (Table II). The
CD4+ clones could secrete IFN- when stimulated
with T2 cells pulsed with m9-27 peptide but not tumor cells. Most
clones were also tested for their ability to secrete cytokines other
than IFN-. All 24 clones tested (6 from PBL-65, 9 from PBL-66, and 9
from PBL-67) specifically secreted IFN- and GM-CSF but not TNF-,
IL-2, IL-4, or IL-10 in response to m9-27 peptide-pulsed T2 cells (data
not shown).
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. The first group of clones (23 of the
34 clones tested), represented by clone 66-63, were
CD8+ T cells, lysed HLA-A2+
MART-1+ targets, and secreted IFN- when
stimulated by m9-27 peptide-pulsed T2 cells or
HLA-A2+ melanoma cell lines. The ability of most
of these clones to lyse targets and secrete cytokine was generally
comparable to the MART-1-reactive TIL 1235. One of the 34 clones tested
(clone 66-36) was CD8+ and lysed
HLA-A2+ MART-1+ targets but
secreted little or no IFN- when stimulated by m9-27 peptide-pulsed
T2 cells or HLA-A2+ melanoma cell lines. The
third group of clones (6 of the 34 clones tested), represented by clone
67-65, were CD8+ and secreted IFN- when
stimulated by m9-27 peptide-pulsed T2 cells or
HLA-A2+ melanoma cell lines but weakly lysed
HLA-A2+ MART-1+ targets.
The fourth group of clones (4 of the 34 clones tested), represented by
clone 65-46, were CD4+ and secreted IFN- when
stimulated by m9-27 peptide-pulsed T2 cells but did not lyse
HLA-A2+ MART-1+ targets. To
insure that the properties observed in these clones were due to a
single clone and not mixed clones, the clonality of a small group of
clones was tested by determining their TCRBV usage with a panel of
TCRBV subfamily-specific primers. Of the 13 clones tested, 10 expressed
only the introduced BV7 gene and one other TCRBV subfamily, indicating
that most of the T cell lines were clonal (data not shown). These
results indicate that MART-1-reactive clones obtained by transducing
normal donor PBL with the TIL 5 TCR were heterogenous with respect to
their ability to lyse and specifically secrete cytokines when cultured
with MART-1+ cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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when
stimulated by very low concentrations of m9-27 peptide pulsed onto T2
cells. These gene-modified T cells capable of recognizing melanoma Ags
thus provide an alternative approach for treating patients with
metastatic melanoma.
Suprisingly, CD4+ clones were also isolated from
a transduced PBL culture that recognized T2 cells pulsed with the m9-27
peptide in cytokine release assays. Recognition occurred in the absence
of CD4 coreceptor interaction with the presenting MHC molecule, because
CD4 cannot bind to HLA-A2.1 (33). The interaction between a TCR and the
peptide-MHC complex is thought to be a low-affinity interaction (34).
CD4 and CD8 facilitate the interaction between a T cell and an APC by
binding to class II or class I MHC molecules, respectively. This is
thought to occur before the TCR/peptide-MHC interaction, increasing the
overall avidity of the TCR/peptide-MHC complex (35, 36, 37). In addition to
their roles as adhesion molecules, there is a close association between
the cytoplasmic tail of CD4 with p56lck
suggesting that CD4 may also have a role in signal transduction during
T cell activation (38, 39). The cytoplasmic tail of the CD8 -chain
also associates with p56lck, and a role in
signal transduction has also been suggested for CD8 (40, 41). The
recognition of m9-27 peptide-pulsed T2 cells by transduced
CD4+ clones demonstrated that the clone 5 cells
had a TCR with sufficient affinity to transduce an activation signal
without the interaction of CD8 coreceptors with HLA-A2. However, the
clone 5 TCR, when expressed in Jurkat cells or normal
CD4+ T cells, was unable to recognize melanoma
cells. This suggests that the high levels of m9-27 peptide present on
T2 cells can overcome the low-affinity of a TCR leading to T cell
activation. We and others have isolated CD8-independent T cell clones
that recognized human melanoma cells in a class I-restricted manner
(M.I.N., unpublished observations) (42, 43, 44). The TCR genes from these
CD8-independent T cell clones likely encode high-affinity TCR, which
would be better candidates for gene transfer.
A potential advantage of transferring a specifically reactive bulk transduced PBL culture to cancer patients is that CD8+ and CD4+ clones expressing different levels of the transferred TCR can have different functional characteristics. The CD4+ clones might augment the activity of CD8+ clones by providing stimulatory cytokines such as IL-2 at the tumor site. However, the relative frequency of lytic CD8+ clones and reactive CD4+ clones in the transduced bulk PBL cultures will be variable and their reactivity may be low. An alternate treatment strategy would be to isolate T cell clones with defined reactivities from these bulk transduced PBL cultures. Tumor-reactive clones from the transduced bulk cultures can be expanded to numbers suitable for adoptive transfer (1010 cells) using the procedures described (22). Clones or pools of these clones could be adoptively transferred into melanoma patients. The transfer of tumor-reactive CD4+ clones with tumor-reactive CD8+ clones may be more effective than transferring the CD8+ clones alone.
The HLA-A2-restricted TCR used in this study could enable us to treat
50% of all melanoma patients because 50% of all melanoma
patients express HLA-A2 (45). However, there are no HLA class II
alleles expressed by >34% of melanoma patients (46). Furthermore,
most melanoma-reactive CD4+ T cells recognize
only their autologous tumors rather than shared Ags. The capacity to
produce HLA-A2-restricted CD4+ T cells that
recognize shared melanoma Ags could enable us to provide T cell help to
more patients than we could treat with MHC class II-restricted
cells.
Because of the unique CDR3 sequences of the clone 5 TCR -and
ß-chains, cPCR can be used to follow adoptively transferred PBL
clones to examine their persistence in the peripheral blood of patients
(47). Biopsies or fine needle aspirates of accessible tumor lesions
could also allow the trafficking of TCR gene-modified T cells to tumors
to be assessed. Because individual transduced PBL clones express a
second unique TCR, the CDR3 region of this TCR will allow individual
transduced clones in a pool of adoptively transferred clones to be
monitored. These studies could provide new insights into the fate of
adoptively transferred T cells. Previous trafficking studies of
adoptively transferred CTL have relied upon the transfer of
radiolabeled cells and therefore were imprecise (48).
A number of other potential applications of TCR gene transfer to PBL are under investigation. T cell clones that are specific for a number of different TAAs have been isolated and a panel of retroviral vectors encoding the TAA-specific TCR from these clones is being assembled. We envision using multiple viruses to transduce separate PBL cultures from a melanoma patient to generate cultures with anti-tumor reactivity against different TAA. Adoptive transfer of CTL pools recognizing multiple TAA could circumvent in vivo immunoselection of tumor cells lacking expression of a single target TAA, which has been reported to be a mechanism of tumor escape from cellular immunotherapies (49). It is conceivable that a single PBL culture could be sequentially transduced with two or more TCR to generate clones expressing multiple tumor-Ag-specific TCR. We are also investigating the transfer of TAA-specific TCR into T cell clones that already have an existing anti-tumor reactivity. We can routinely generate MART-1 m9-27-reactive CTL by multiple rounds of in vitro peptide stimulation of melanoma patient PBMC in the majority of patients tested (50, 51). Bispecific clones could also be generated by transducing T cell clones that target the tumor vasculature, such as clones specific for the vascular endothelium growth factor receptor. These studies could provide unique opportunities to study T cell biology. And finally, the transfer of TCR genes to human PBL might also be used to identify new TAA. Where a CTL clone with a unique reactivity has been isolated but cannot be expanded to sufficiently high numbers to permit the screening of a cDNA library, transfer of the clone TCR to PBL could provide an inexhaustible supply of T cells for library screening.
Human clinical trials with adoptive transfer of TCR gene-modified PBL clones can evaluate the effectiveness of these approaches for the treatment of patients with metastatic melanoma. The success of retroviral transduction in three PBL tested in this study demonstrates that this technique would provide a source of anti-tumor CTL for immunotherapy protocols. As CTL that recognize TAA in other tumor histologies are identified, the TCR genes from these T cells could potentially be used for the therapy of other cancers. These approaches could also be applied to the treatment of viral infections.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: TAA, tumor associated Ag; CDR3, third complementarity determining region; cPCR, competitive PCR; TCRBV, TCR Vß; TIL, tumor infiltrating lymphocyte.
Received for publication February 1, 1999. Accepted for publication April 14, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3 domain of HLA-A2. Nature 345:41.[Medline]
. Cancer Immunol. Immunother. 37:187.[Medline]
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Z. Sebestyen, E. Schooten, T. Sals, I. Zaldivar, E. San Jose, B. Alarcon, S. Bobisse, A. Rosato, J. Szollosi, J. W. Gratama, et al. Human TCR That Incorporate CD3{zeta} Induce Highly Preferred Pairing between TCR{alpha} and {beta} Chains following Gene Transfer J. Immunol., June 1, 2008; 180(11): 7736 - 7746. [Abstract] [Full Text] [PDF]
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Y. Hou, B. Kavanagh, and L. Fong Distinct CD8+ T Cell Repertoires Primed with Agonist and Native Peptides Derived from a Tumor-Associated Antigen J. Immunol., February 1, 2008; 180(3): 1526 - 1534. [Abstract] [Full Text] [PDF]
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 A. Jorritsma, R. Gomez-Eerland, M. Dokter, W. van de Kasteele, Y. M. Zoet, I. I. N. Doxiadis, N. Rufer, P. Romero, R. A. Morgan, T. N. M. Schumacher, et al. Selecting highly affine and well-expressed TCRs for gene therapy of melanoma Blood, November 15, 2007; 110(10): 3564 - 3572. [Abstract] [Full Text] [PDF]
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S. Thomas, S.-A. Xue, M. Cesco-Gaspere, E. San Jose, D. P. Hart, V. Wong, R. Debets, B. Alarcon, E. Morris, and H. J. Stauss Targeting the Wilms Tumor Antigen 1 by TCR Gene Transfer: TCR Variants Improve Tetramer Binding but Not the Function of Gene Modified Human T Cells J. Immunol., November 1, 2007; 179(9): 5803 - 5810. [Abstract] [Full Text] [PDF]
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 W Yang, E. Beaudoin, L Lu, R. Du Pasquier, M. Kuroda, R. Willemsen, I. Koralnik, and R. Junghans Chimeric immune receptors (CIRs) specific to JC virus for immunotherapy in progressive multifocal leukoencephalopathy (PML) Int. Immunol., September 1, 2007; 19(9): 1083 - 1093. [Abstract] [Full Text] [PDF]
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J. Kuball, M. L. Dossett, M. Wolfl, W. Y. Ho, R.-H. Voss, C. Fowler, and P. D. Greenberg Facilitating matched pairing and expression of TCR chains introduced into human T cells Blood, March 15, 2007; 109(6): 2331 - 2338. [Abstract] [Full Text] [PDF]
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M. H. M. Heemskerk, R. S. Hagedoorn, M. A. W. G. van der Hoorn, L. T. van der Veken, M. Hoogeboom, M. G. D. Kester, R. Willemze, and J. H. F. Falkenburg Efficiency of T-cell receptor expression in dual-specific T cells is controlled by the intrinsic qualities of the TCR chains within the TCR-CD3 complex Blood, January 1, 2007; 109(1): 235 - 243. [Abstract] [Full Text] [PDF]
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G. E. Lyons, T. Moore, N. Brasic, M. Li, J. J. Roszkowski, and M. I. Nishimura Influence of Human CD8 on Antigen Recognition by T-Cell Receptor-Transduced Cells Cancer Res., December 1, 2006; 66(23): 11455 - 11461. [Abstract] [Full Text] [PDF]
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M. A. de Witte, M. Coccoris, M. C. Wolkers, M. D. van den Boom, E. M. Mesman, J.-Y. Song, M. van der Valk, J. B. A. G. Haanen, and T. N. M. Schumacher Targeting self-antigens through allogeneic TCR gene transfer Blood, August 1, 2006; 108(3): 870 - 877. [Abstract] [Full Text] [PDF]
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N. Schaft, B. Lankiewicz, J. Drexhage, C. Berrevoets, D. J. Moss, V. Levitsky, M. Bonneville, S. P. Lee, A. J. McMichael, J.-W. Gratama, et al. T cell re-targeting to EBV antigens following TCR gene transfer: CD28-containing receptors mediate enhanced antigen-specific IFN{gamma} production Int. Immunol., April 1, 2006; 18(4): 591 - 601. [Abstract] [Full Text] [PDF]
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L. T. van der Veken, R. S. Hagedoorn, M. M. van Loenen, R. Willemze, J.H. F. Falkenburg, and M. H.M. Heemskerk {alpha}{beta} T-Cell Receptor Engineered {gamma}{delta} T Cells Mediate Effective Antileukemic Reactivity. Cancer Res., March 15, 2006; 66(6): 3331 - 3337. [Abstract] [Full Text] [PDF]
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 L. Duval, H. Schmidt, K. Kaltoft, K. Fode, J. J. Jensen, S. M. Sorensen, M. I. Nishimura, and H. von der Maase Adoptive Transfer of Allogeneic Cytotoxic T Lymphocytes Equipped with a HLA-A2 Restricted MART-1 T-Cell Receptor: A Phase I Trial in Metastatic Melanoma Clin. Cancer Res., February 15, 2006; 12(4): 1229 - 1236. [Abstract] [Full Text] [PDF]
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S.-A. Xue, L. Gao, D. Hart, R. Gillmore, W. Qasim, A. Thrasher, J. Apperley, B. Engels, W. Uckert, E. Morris, et al. Elimination of human leukemia cells in NOD/SCID mice by WT1-TCR gene-transduced human T cells Blood, November 1, 2005; 106(9): 3062 - 3067. [Abstract] [Full Text] [PDF]
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T. Zhang, B. A. Lemoi, and C. L. Sentman Chimeric NK-receptor-bearing T cells mediate antitumor immunotherapy Blood, September 1, 2005; 106(5): 1544 - 1551. [Abstract] [Full Text] [PDF]
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T. Tsuji, M. Yasukawa, J. Matsuzaki, T. Ohkuri, K. Chamoto, D. Wakita, T. Azuma, H. Niiya, H. Miyoshi, K. Kuzushima, et al. Generation of tumor-specific, HLA class I-restricted human Th1 and Tc1 cells by cell engineering with tumor peptide-specific T-cell receptor genes Blood, July 15, 2005; 106(2): 470 - 476. [Abstract] [Full Text] [PDF]
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R. A. Willemsen, C. Ronteltap, P. Chames, R. Debets, and R. L. H. Bolhuis T Cell Retargeting with MHC Class I-Restricted Antibodies: The CD28 Costimulatory Domain Enhances Antigen-Specific Cytotoxicity and Cytokine Production J. Immunol., June 15, 2005; 174(12): 7853 - 7858. [Abstract] [Full Text] [PDF]
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 E. C. Morris, A. Tsallios, G. M. Bendle, S.-a. Xue, and H. J. Stauss A critical role of T cell antigen receptor-transduced MHC class I-restricted helper T cells in tumor protection PNAS, May 31, 2005; 102(22): 7934 - 7939. [Abstract] [Full Text] [PDF]
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J. Charo, S. E. Finkelstein, N. Grewal, N. P. Restifo, P. F. Robbins, and S. A. Rosenberg Bcl-2 Overexpression Enhances Tumor-Specific T-Cell Survival Cancer Res., March 1, 2005; 65(5): 2001 - 2008. [Abstract] [Full Text] [PDF]
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J. J. Roszkowski, G. E. Lyons, W. M. Kast, C. Yee, K. Van Besien, and M. I. Nishimura Simultaneous Generation of CD8+ and CD4+ Melanoma-Reactive T Cells by Retroviral-Mediated Transfer of a Single T-Cell Receptor Cancer Res., February 15, 2005; 65(4): 1570 - 1576. [Abstract] [Full Text] [PDF]
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 M. H.M. Heemskerk, M. Hoogeboom, R. Hagedoorn, M. G.D. Kester, R. Willemze, and J.H. F. Falkenburg Reprogramming of Virus-specific T Cells into Leukemia-reactive T Cells Using T Cell Receptor Gene Transfer J. Exp. Med., April 5, 2004; 199(7): 885 - 894. [Abstract] [Full Text] [PDF]
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M. H. M. Heemskerk, M. Hoogeboom, R. A. de Paus, M. G. D. Kester, M. A. W. G. van der Hoorn, E. Goulmy, R. Willemze, and J. H. F. Falkenburg Redirection of antileukemic reactivity of peripheral T lymphocytes using gene transfer of minor histocompatibility antigen HA-2-specific T-cell receptor complexes expressing a conserved alpha joining region Blood, November 15, 2003; 102(10): 3530 - 3540. [Abstract] [Full Text] [PDF]
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R. A. Morgan, M. E. Dudley, Y. Y. L. Yu, Z. Zheng, P. F. Robbins, M. R. Theoret, J. R. Wunderlich, M. S. Hughes, N. P. Restifo, and S. A. Rosenberg High Efficiency TCR Gene Transfer into Primary Human Lymphocytes Affords Avid Recognition of Melanoma Tumor Antigen Glycoprotein 100 and Does Not Alter the Recognition of Autologous Melanoma Antigens J. Immunol., September 15, 2003; 171(6): 3287 - 3295. [Abstract] [Full Text] [PDF]
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H. Tahara, K. Fujio, Y. Araki, K. Setoguchi, Y. Misaki, T. Kitamura, and K. Yamamoto Reconstitution of CD8+ T Cells by Retroviral Transfer of the TCR {alpha}{beta}-Chain Genes Isolated from a Clonally Expanded P815-Infiltrating Lymphocyte J. Immunol., August 15, 2003; 171(4): 2154 - 2160. [Abstract] [Full Text] [PDF]
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K.-M. Lee, S. Bhawan, T. Majima, H. Wei, M. I. Nishimura, H. Yagita, and V. Kumar Cutting Edge: The NK Cell Receptor 2B4 Augments Antigen-Specific T Cell Cytotoxicity Through CD48 Ligation on Neighboring T Cells J. Immunol., May 15, 2003; 170(10): 4881 - 4885. [Abstract] [Full Text] [PDF]
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J. J. Roszkowski, D. C. Yu, M. P. Rubinstein, M. D. McKee, D. J. Cole, and M. I. Nishimura CD8-Independent Tumor Cell Recognition Is a Property of the T Cell Receptor and Not the T Cell J. Immunol., March 1, 2003; 170(5): 2582 - 2589. [Abstract] [Full Text] [PDF]
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N. Schaft, R. A. Willemsen, J. de Vries, B. Lankiewicz, B. W. L. Essers, J.-W. Gratama, C. G. Figdor, R. L. H. Bolhuis, R. Debets, and G. J. Adema Peptide Fine Specificity of Anti-Glycoprotein 100 CTL Is Preserved Following Transfer of Engineered TCR{alpha}{beta} Genes Into Primary Human T Lymphocytes J. Immunol., February 15, 2003; 170(4): 2186 - 2194. [Abstract] [Full Text] [PDF]
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M. P. Rubinstein, A. N. Kadima, M. L. Salem, C. L. Nguyen, W. E. Gillanders, M. I. Nishimura, and D. J. Cole Transfer of TCR Genes into Mature T Cells Is Accompanied by the Maintenance of Parental T Cell Avidity J. Immunol., February 1, 2003; 170(3): 1209 - 1217. [Abstract] [Full Text] [PDF]
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T.-H. Yang, M. Lovatt, M. Merkenschlager, and H. J. Stauss Comparison of the frequency of peptide-specific cytotoxic T lymphocytes restricted by self- and allo-MHC following in vitro T cell priming Int. Immunol., November 1, 2002; 14(11): 1283 - 1290. [Abstract] [Full Text] [PDF]
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L. Yang, X.-F. Qin, D. Baltimore, and L. Van Parijs Generation of functional antigen-specific T cells in defined genetic backgrounds by retrovirus-mediated expression of TCR cDNAs in hematopoietic precursor cells PNAS, April 30, 2002; 99(9): 6204 - 6209. [Abstract] [Full Text] [PDF]
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J. V. Brawley and P. Concannon Complementarity-Determining Region 1 Sequence Requirements Drive Limited V{alpha} Usage in Response to Influenza Hemagglutinin 307-319 Peptide J. Immunol., April 15, 2002; 168(8): 3894 - 3901. [Abstract] [Full Text] [PDF]
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H. W. H. G. Kessels, M. D. van den Boom, H. Spits, E. Hooijberg, and T. N. M. Schumacher Changing T cell specificity by retroviral T cell receptor display PNAS, December 19, 2000; 97(26): 14578 - 14583. [Abstract] [Full Text] [PDF]
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M. Migliaccio, M. Amacker, T. Just, P. Reichenbach, D. Valmori, J.-C. Cerottini, P. Romero, and M. Nabholz Ectopic Human Telomerase Catalytic Subunit Expression Maintains Telomere Length But Is Not Sufficient for CD8+ T Lymphocyte Immortalization J. Immunol., November 1, 2000; 165(9): 4978 - 4984. [Abstract] [Full Text] [PDF]
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 L. J. N. Cooper, M. Kalos, D. A. Lewinsohn, S. R. Riddell, and P. D. Greenberg Transfer of Specificity for Human Immunodeficiency Virus Type 1 into Primary Human T Lymphocytes by Introduction of T-Cell Receptor Genes J. Virol., September 1, 2000; 74(17): 8207 - 8212. [Abstract] [Full Text]
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 A. Ribas, L. H. Butterfield, and J. S. Economou Genetic Immunotherapy for Cancer Oncologist, April 1, 2000; 5(2): 87 - 98. [Abstract] [Full Text]
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X. Liu, E. A. Peralta, J. D. I. Ellenhorn, and D. J. Diamond Targeting of Human p53-overexpressing Tumor Cells by an HLA A*0201-restricted Murine T-Cell Receptor Expressed in Jurkat T Cells Cancer Res., February 1, 2000; 60(3): 693 - 701. [Abstract] [Full Text]
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M. I. Nishimura, D. Avichezer, M. C. Custer, C. S. Lee, C. Chen, M. R. Parkhurst, R. A. Diamond, P. F. Robbins, D. J. Schwartzentruber, and S. A. Rosenberg MHC Class I-restricted Recognition of a Melanoma Antigen by a Human CD4+ Tumor Infiltrating Lymphocyte Cancer Res., December 1, 1999; 59(24): 6230 - 6238. [Abstract] [Full Text] [PDF]
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packaging signal. Arrows denote transcription start sites.