multiple lymphocyte types including NK cell precursors and T lymphocytes, and they exhibited HLA-independent lysis of tumor cells in vitro. Many of the foundations for large scale human lymphocyte cultures were established in these trials, including the formulation of serum free media, methods for sterilely handling and processing large volumes of cells, and safety and efficacy tests for human lymphocyte infusions for clinical trials 2. However, LAK cells with high dose IL-2 were not shown to be effective in a randomized clinical trial when compared to IL-2 alone 3.In contrast to the non-specific activity of LAK cells, the cultures derived from some tumor infiltrating lymphocytes (TIL) that were expanded in vitro in IL-2 demonstrated marked tumor specificity. This phenomenon was initially described with lymphocytes from immunogenic transplantable tumors of mice 4, and then characterized in TIL derived from resected human cancers of several histologies 5–7. Although TIL cultures could sometimes be expanded from tumors of common epithelial origin, these cultures rarely demonstrated specific tumor activity.In contrast, melanoma TIL have been reliably generated using two related methods in the Surgery Branch, and tumor specific activity was detected by lysis or cytokine release in about 70% of cultures in two separate large series of samples 7,8. The study of the antigen reactive lymphocytes that infiltrate melanoma metastases has informed and driven many of the Surgery Branch clinical cell transfer efforts.Initial clinical efforts with TIL were summarized in a study using autologous TIL plus IL-2 in the treatment of 86 patients with metastatic melanoma 9. In that stud
y TIL and high dose IL-2were administered in two cycles separated by approximately 2 weeks, constituting one treatment course. Six weeks after treatment, all known sites of disease were evaluated. The overall objective response rate in those patients was 34%, although many of the responses were of short duration. There was no significant difference in the objective response rate in patients whose therapy with high-dose IL-2 had failed (32%) compared with patients not previously treated with IL-2 (34%). Since patients who recurred after initially responding to IL-2 do not
respond to additional cycles of IL-210 the response of patients who received TIL plus IL-2
strongly implied that the anti-tumor response was mediated by the TIL cells. These results
illustrated the potential value of immune lymphocytes for the treatment of patients with
melanoma, and laid the foundation for many of the cell transfer studies that followed in the
Surgery Branch and at other institutions.
Retrospective analyses of treatment characteristics of the infused cells and the patients’ clinical
outcomes revealed several strong correlations. The frequency of response to treatment was
greater in patients who were treated with TIL from younger cultures (P = .0001) and TIL with
shorter doubling times (P = .03). Another strong correlation was noted between response and
TIL that exhibited higher lysis against autologous tumor targets (P = .0008). Probably related
to this issue of tumor specific recognition, patients who received TIL generated from
subcutaneous tumor deposits had higher response rates (49%) compared with those receiving
TIL from lymph nodes (17%; P = .006). Some subsequent research efforts and clinical trials
in the Surgery Branch have focused on the issues defined by these correlations: improved anti-
tumor specificity of the treatment TIL, or improved growth and expansion conditions to yield
optimized early growth of TIL that can engraft, persist, and mediate anti-tumor effects in
vivo .
TIL cells from responding patients were used for molecular identification of tumor rejection
antigens by cDNA expression cloning. One of the first antigens cloned was the melanoma
antigen recognized by T cells (MART)-1 gene, which was cloned using both transient and
stable expression systems 11. MART-1 is a transmembrane protein whose expression is
restricted to melanoma cells and normal melanin producing cells, but no other normal tissues
or tumor histologies. MART-1 is thus a melanocyte lineage-specific protein and a widely
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shared melanoma antigen. In one study, the presence of gp100 reactive cells was significantly
correlated with patient response (p=0.005) suggesting that gp100 may be useful for the
development of immunotherapies for patients with melanoma 12. Currently, many genes
expressed by tumors have been identified whose normally transcribed or mutated gene products
are recognized by T cells, and which are candidates for immune targetting 13.
The identification of the gp100 gene product and its HLA-A2 restricted epitopes as targets for
the immunotherapy opened new approaches to cell transfer therapy. The gp100:209–217
epitope was shown to be a relatively poor immunogen in humans, but its immunogenicity was
improved by the modification of the nine amino acid peptide sequence to enhance its HLA-
A2 binding affinity without substantially impacting the T-cell contact surface of the HLA-
peptide-T cell receptor interface 14. Using a nine amino acid peptide with a methionine
substitution for a threonine at the second position, a T cell epitope with a better in vitro and in
vivo immunizing profile was identified. Patients were immunized as part of clinical trials, both
in the adjuvant setting and for metastatic disease, using the modified 209-2M peptide
vaccine 15. Most patients developed high levels of gp100-specific, tumor reactive CTL
precursors in their peripheral circulation after vaccination. However, patients with metastatic
disease rarely experienced clinical remission due to vaccination alone, and highly immunized
patients recurred with disease in adjuvant vaccine studies 16. However, the presence of large
numbers of highly active, highly avid anti-tumor T cell precursors in the PBL enabled the
design and implementation of new clinical trials to evaluate their efficacy in adoptive cell
transfer experiments. Several methods were devised for the isolation, expansion, and activation
of highly avid tumor-specific lymphocytes from PBL precursors, and their administration at
high numbers to autologous patients.Cloned PBL and IVS PBL A phase I study of the adoptive transfer of cloned melanoma antigen-specific T lymphocytes for therapy of patients with advanced melanoma was undertaken in the Surgery Branch,
NCI 17. In that study, clones were derived from PBL or TIL of patients who had received prior
immunization with 209-2M. In response to their cognate antigens, clones used for treatment
secreted large amounts of interferon-gamma (IFNg) and GM-CSF, lesser amounts of IL-2 and
tumor necrosis factor alpha (TNFa), and little or no IL-4 and IL-10. Clones also demonstrated
recognition of HLA-matched melanomas using cytokine secretion and lysis assays. Eleven
patients received 2 cycles of cells alone and then were randomized between two schedules of
IL-2: low dose (125,000 IU/kg subcutaneously daily for 12 days) or high dose (720,000 IU/kg
intravenously every 8hr for 4 days); one additional patient received two cycles of cloned cells
alone and was discharged without finishing the treatment course. An average of 1 × 1010 cells
was administered per cycle. For some patients, peripheral blood samples were analyzed for
persistence of transferred cells by T-cell receptor-specific PCR. Although the transferred cells
were detectable at 1hr after transfer, they rapidly declined to undetectable levels by 2 weeks.
One minor response and one mixed response were observed (both in the high-dose IL-2 arm),
but no patient achieved an objective clinical response by RECIST criteria.
A similar study was undertaken by Yee et al.18 with similar results. T cell clones targeting the
tumor-associated antigens, MART1 and gp100, were selected and expanded in vitro for the
treatment of patients with metastatic melanoma. Four infusions of autologous T cell clones
were administered to 10 patients, the first without IL-2 and subsequent infusions with low-
dose IL-2. The transferred T cell clones persisted while the low dose IL-2 administration was
ongoing (approximately 15 days) but rapidly disappeared from the peripheral circulation
thereafter. Overall, the regression of individual metastases, and minor, mixed or stable
responses in individual patients were reported, but no objective clinical responses were seen
by RECIST criteria.
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Together, these reports on the transfer of cloned antigen specific T cells demonstrated the safety and feasibility of this approach for patients with cancer. However, the lack of clinical effectiveness of the cloned T lymphocytes suggested that transfer of different or additional cell types or that modulation of the recipient host environment was required for successful therapy.The need to grow cloned lymphocytes in vitro for extended times, and the single specificity of the antigen receptor were both potential reasons for the lack of effectiveness of these cloned cell protocols.One of the particularly impressive deficits of the cloned lymphocytes in vivo was their rapid disappearance from the peripheral circulation after transfer, despite the transfer of large numbers of cells. We hypothesized that competition for homeostatic cytokines such as IL-7 or IL-15 could be limiting the persistence of the transferred tumor reactive clones, and that elimination of the endogenous host repertoire might prolong persistence of the transferred cells and enhance anti-tumor efficacy. To investigate this hypothesis, we initiated a clinical trial using a non-myeloablative, lymphodepleting chemotherapy in combination with adoptive immunotherapy in patients with metastatic melanoma 19. Initially, a dose escalation phase was undertaken to evaluate the tolerance to the non-myeloablative chemotherapy in
combination with IL-2. The chemotherapy conditioning schedule induced transient lymphopenia and consisted of cyclophosphamide (30 or 60 mg/kg per day for 2 days) followed by fludarabine (25 mg/m2 per day for 5 days). Immunotherapy for all patients consisted of in vitro expanded,tumor-reactive, autologous T-cell clones selected for highly avid recognition of melanoma antigens. Cohorts of three to six patients received either no interleukin (IL)-2, low-dose IL-2(72,000 IU/kg intravenously three times a day to a maximum of 15 doses), or high-dose IL-2(720,000 IU/kg intravenously three times a day for a maximum of 12 doses). The toxicities associated with this treatment were transient and included neutropenia and thrombocytopenia that resolved in all patients. The toxicities associated with this dose escalation chemotherapy regimen are shown in Table 1. No patient exhibited an objective clinical response to treatment,although five patients demonstrated mixed responses or transient shrinkage of some metastatic
deposits. Surprisingly, high dose intravenous IL-2 was better tolerated by patients after
chemotherapy than during previous immunotherapy cycles without chemotherapy, suggesting
that some toxicities of IL-2 were mediated by cytokines secreted by lymphocytes secondary
to activation by IL-2.
Powell et al.20 and Mackensen et al.21 used different methods to overcome the limitations of
cloning techniques to generate large numbers of tumor antigen reactive lymphocytes from PBL
for adoptive cell transfer protocols. Powell et al. assessed the capacity of vaccine-induced
PBMC to mediate tumor regression after transfer to patients receiving the maximum dose of
non-myeloablative preparative chemotherapy discussed above along with high dose IL-2.
reactive materials studiesAutologous PBMC from nine gp100-vaccinated patients with metastatic melanoma were
stimulated ex vivo with the 209-2M peptide and transferred in combination with high-dose
IL-2 and a peptide cancer vaccine. Transferred PBMC contained highly avid, gp100:209-217
peptide-reactive CD8+ T cells. Unlike the transferred cloned cells, many of the bulk PBL
populations caused a significant lymphocytosis in the patients, with peak counts approximately
one week after transfer. Tumor-antigen reactive CD8+ T cells persisted at high levels in the
blood of all patients and demonstrated IFN-gamma secretion when restimulated in vitro. Two
of the nine patients demonstrated some evidence of biological activity and melanocyte-directed
autoimmunity; however, no patient experienced an objective clinical response.
A similar phase I study was conducted by Mackensen et al. using multiply restimulated PBL
cultures that reacted with the MART-1/MELAN-A antigen in eleven HLA-A2+ patients with
metastatic melanoma 21. Patients in this study received at least three infusions of MART-1
reactive CTL cultures that were generated by repetitive stimulation with autologous dendritic
cells pulsed with MART-1 derived peptide. Patients also received a 6-day course of low-dose
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IL-2 after each cell infusion. There were no serious adverse events reported. This study group included one patient with an objective partial response and one patient with a complete response, anb the transferred cells persisted at low levels in patients’ circulating blood for up to 2 weeks. Overall, these studies demonstrated the feasibility and safety of using peripheral blood derived lymphocytes for adoptive cell transfer, but suggested that the cultures induced after multiple in vitro  restimulations used by Mackensen’s group, or the vaccine induced populations of cells used by Powell et al, lacked sufficient potency in adoptive immunotherapy even when transferred into lymphodepleted hosts.TIL with a lymphodepleting preparative regimen Several methods had been established for the generation of tumor antigen-reactive lymphocyte cultures and evaluated in clinical trials. However, highly cultured, highly expanded cloned lymphocytes lacked potency in vivo. Although bulk TIL could mediate objective clinical responses, they were often of short duration and the bulk TIL were logistically difficult to grow 9. Because it had been established that a non-myeloablative conditioning regimen could be safely administered in conjunction with adoptive T-cell transfer and IL-2 in patients with metastatic melanoma, we next evaluated whether highly selected, tumor antigen reactive, bulk TIL populations that were rapidly expanded could mediate anti-tumor responses. In an initial cohort of 13 patients 22, our results were markedly different than those seen after administration of cloned lymphocyte cultures. This approach resulted in the persistent repopulation of anti-tumor T cells in the cancer patients, with prolife
ration of functionally active transferred cells in vivo  and traffic to tumor sites. This led to regression of the metastatic melanoma in six of 13 patients as well as the onset of autoimmune melanocyte destruction in some patients.This initial cohort of patients was followed with accrual of additional patients. We reported the combination of lymphodepleting chemotherapy followed by the adoptive transfer of autologous tumor reactive lymphocytes for the treatment of 35 patients with refractory metastatic melanoma 23. All but one of these patients had disease that was refractory to
treatment with high-dose IL -2 and 18 had progressive disease after chemotherapy. Patients
underwent non-myeloablative lymphodepleting conditioning with cyclophosphamide and
fludarabine, followed by cell infusion with autologous tumor-reactive, rapidly expanded TIL
cultures and high-dose IL-2 therapy. Eighteen treated patients (51%) experienced objective
clinical responses by RECIST criteria, including three complete responses that are ongoing
more than three years after treatment. Sites of regression included metastases to lung, liver,
lymph nodes, brain, and cutaneous and subcutaneous tissues. Although the toxicities of
treatment were acute and severe, including the expected toxicities of high-dose IL-2 therapy
and the hematologic suppression of chemotherapy, most toxicities were transient and resolved
within two weeks. These results suggested that lymphodepleting chemotherapy followed by
the transfer of highly avid anti-tumor lymphocytes could mediate significant tumor regression
in heavily pretreated patients with IL-2 refractory metastatic melanoma. Table 2 shows the
patients who responded to this treatment, along with the duration of their response as of May,
2007, and the initial sites of disease.
A notable immunologic correlate of successful treatment in many patients was the presence of
the transferred cells in the peripheral blood for extended durations. Figure 1 shows a FACS
analysis of the skewed profile of peripheral blood of several patients who responded to
treatment, and the persistence of a dominant anti-tumor clonotype that was visualized by its T
cell receptor V beta or tetramer profile. To quantify this relationship more exactly, TCR beta-
chain V region gene product expressions was analyzed from samples obtained from 25 patients
treated with TIL 24. Sequence analysis demonstrated that there was a significant correlation
between tumor regression and the degree of persistence in peripheral blood of adoptively
transferred T cell clones, suggesting that inadequate T cell persistence might have represented
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a major factor limiting responses to adoptive immunotherapy in that protocol. To investigate
this idea, Powell et al studied the transition from activated, cultured T cells to memory cells
after transfer in serial blood draws from responding patients 25. This study showed that the
melanoma antigen-specific CD8+ T cells rapidly up-regulated their IL-7R-alpha in vivo.
Furthermore, although the total number of tumor antigen-specific T-cells decreased two weeks
after transfer, stable numbers of CD27+ CD28+ cells were maintained, and these cells formed
the long-lived memory pool.
These findings, which suggested that the proliferative potential of transferred T cells may play
a role in clinical responses, led to studies investigating the role of telomere length as well as
phenotypic markers expressed on the administered TIL 26. After evaluating all available
samples from patients who were treated with rapidly expanded TIL following non-
myeloablative chemotherapy, it was found that TIL administered to patients who experienced
objective clinical responses possessed a mean telomere length that was significantly longer
than TIL given to non-responding patients (p < 0.01). Furthermore, individual TIL-derived T
cell clonotypes that persisted in vivo following adoptive cell transfer possessed telomeres that
were longer than telomeres of T cell clonotypes that failed to persist (p < 0.001). These results
were supported by studies suggesting that higher CD28 expression was also associated with
TIL that caused objective responses. Together these results suggest that TIL with longer
telomeres, higher expression of the costimulatory molecule CD28, and greater proliferative
potential will have a greater likelihood of persisting in vivo and mediating an objective clinical
response.
More recently we have attempted to improve on the response rate of cell transfer therapy by
increasing the lymphodepleting preparative regimen. Murine models demonstrated that the
intensity of lymphodepletion was directly related to the effectiveness of cell transfer therapy.
Therefore, we investigated the addition of 200 cGy total body irradiation together with CD34
+ hematopoeitic stem cell support to the previously reported cell transfer preparative regimen
that consisted of cyclophosphamide and fludarabine. Twenty-five patients with refractory
metastatic melanoma received autologous, tumor reactive lymphocytes following the increased
intensity preparative regimen, and thirteen (52%) exhibited an objective response by RECIST
criteria, including two complete responses. Regression of bulky tumors was observed in various
sites, including cutaneous deposits, nodal disease, visceral sites, brain lesions, and bony sites.
Although the toxicities of treatment were acute and often intense, included myelosuppression
and the toxicities of IL-2 administration, they were typically transient and usually resolved
within two weeks; however, one treatment related mortality occurred in this study. Of interest,
when the TIL were administered, the circulating levels of the T cell homeostatic cytokines IL-7
and IL-15 were significantly elevated. As observed in prior cell transfer protocols, some
patients exhibited prolonged persistence of the transferred lymphocytes in their peripheral
blood. These results confirm the effectiveness of cell transfer immunotherapy for patients with
refractory melanoma. Examples of the potency of this treatment to mediate tumor regression
are shown in Figure 2.TCR gene therapy with cell transfer
TIL therapy following lymphodepletion has been effective in causing tumor regression in over
50% of treated patients. However, the method for TIL production is logistically and technically
demanding, and tumor-reactive TIL culture are only derived from approximately half of the
resected melanoma samples intended for patient treatment. An alternate method for the
production of tumor-reactive cells relies on the use of genetic engineering to confer new antigen
specificity to patients’ cells. To achieve this clinically, the genes for the alpha and beta chains
of a highly reactive anti-MART-1 T-cell receptor were isolated from a T-lymphocyte that
mediated an objective clinical response and in vivo tumor regression in an HLA-A2+ patient
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