InTechOpen uses cookies to offer you the best online experience. By continuing to use our site, you agree to our Privacy Policy.

Medicine » Oncology » "Melanoma - Current Clinical Management and Future Therapeutics", book edited by Mandi Murph , ISBN 978-953-51-2036-0, Published: April 1, 2015 under CC BY 3.0 license. © The Author(s).

Chapter 10

Can Redirected T Cells Outsmart Aggressive Melanoma? The Promise and Challenge of Adoptive Cell Therapy

By Jennifer Makalowski and Hinrich Abken
DOI: 10.5772/59255

Article top


T cells used in adoptive cell therapy of melanoma. (A) Tumor infiltrating T cells (TILs) are isolated from melanoma biopsies, selected for reactivity towards melanoma cells, amplified in the presence of IL-2 to clinically relevant numbers and infused to the patient. Alternatively, TILs are expanded without prior selection for melanoma reactivity using a short-term amplification protocol ("young TILs"). (B) T cells from the peripheral blood of melanoma patients are genetically modified by retro- or lentivirus transduction to express a recombinant T cell receptor (TCR) or a chimeric antigen receptor (CAR), specific for a melanoma associated antigen, amplified and administered to the patient.
Figure 1. T cells used in adoptive cell therapy of melanoma. (A) Tumor infiltrating T cells (TILs) are isolated from melanoma biopsies, selected for reactivity towards melanoma cells, amplified in the presence of IL-2 to clinically relevant numbers and infused to the patient. Alternatively, TILs are expanded without prior selection for melanoma reactivity using a short-term amplification protocol ("young TILs"). (B) T cells from the peripheral blood of melanoma patients are genetically modified by retro- or lentivirus transduction to express a recombinant T cell receptor (TCR) or a chimeric antigen receptor (CAR), specific for a melanoma associated antigen, amplified and administered to the patient.
T cells with engineered specificity. T cells physiologically recognize their target by the T cell receptor (TCR) complex which is composed of the TCR α and β chain for recognition and the CD3 chains for signaling. The variable regions of each TCR chain (Vα and Vβ) together bind to the MHC presented antigen, Cα and Cβ represent the constant domains. T cells can be genetically engineered with defined specificity by expression of recombinant TCR αβ chains of known specificity. In contrast to the TCR, the chimeric antigen receptor (CAR) is one polypeptide chain composed of a single chain fragment of variable region (scFv) antibody for antigen recognition, the extracellular spacer domain, a trans-membrane domain and the intracellular CD3ζ ("first generation" CAR), the CD28-CD3ζ ("second generation" CAR), or the CD28-OX40-CD3ζ ("third generation" CAR) signaling chain.
Figure 2. T cells with engineered specificity. T cells physiologically recognize their target by the T cell receptor (TCR) complex which is composed of the TCR α and β chain for recognition and the CD3 chains for signaling. The variable regions of each TCR chain (Vα and Vβ) together bind to the MHC presented antigen, Cα and Cβ represent the constant domains. T cells can be genetically engineered with defined specificity by expression of recombinant TCR αβ chains of known specificity. In contrast to the TCR, the chimeric antigen receptor (CAR) is one polypeptide chain composed of a single chain fragment of variable region (scFv) antibody for antigen recognition, the extracellular spacer domain, a trans-membrane domain and the intracellular CD3ζ ("first generation" CAR), the CD28-CD3ζ ("second generation" CAR), or the CD28-OX40-CD3ζ ("third generation" CAR) signaling chain.

Can Redirected T Cells Outsmart Aggressive Melanoma? The Promise and Challenge of Adoptive Cell Therapy

Jennifer Makalowski1, 2 and Hinrich Abken1, 2

1. Introduction

1.1. The challenge to induce lasting remission in late stage melanoma

In early stages of the disease surgical resection of melanoma lesions is a curative option; a 10-year-survival rate of 75-85% can be achieved in stage I or II of the disease. However, stage III or IV melanoma is associated with low survival rates of less than 1 year upon diagnosis [1]. The poor prognosis in advanced stages of the disease is thought to be particularly due to the properties of melanoma cells to systemically spread into various organs, to form micro-metastases beyond the detection limit of current imaging procedures [2, 3] and to give rise to relapse of the disease. This is even the case after initially complete response to therapy and after more than a decade from initial treatment. Durable remission is so far only achieved in pre-defined patient subsets despite the development of novel drugs and major improvements in therapeutic regimens [4-6]. This unsatisfactory situation is thought to be due to the extraordinary property of melanoma cells to persist in “dormancy” for long periods of time which is associated with their resistance to chemo-and radiotherapy [7-10]. Taken together, durable cure from melanoma requires eliminating single melanoma cells in a highly specific and efficient fashion even in dormant micro-metastatic lesions.

In this situation recruiting the cellular immune defense machinery to detect and destroy individual melanoma cells is a powerful alternative to conventional therapeutic regimens. The hope is sustained by the supportive effect of high dose interleukin-2 (IL-2) [11] and anti-cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) antibody [12] as well as interferon (IFN) α-2b to prolong the disease-free survival even in late stages of the disease. However, the response rate is quite low and frequently not curative over time [13, 14].

A number of strategies for sharpening the immune cell response against melanoma are currently explored, some of these with remarkable success. In particular, the adoptive transfer of tumor infiltrating lymphocytes (TILs), isolated from melanoma biopsies and amplified to therapeutic relevant numbers ex vivo, produced encouraging phase II results [15, 16]. In a further development, patients’ blood T cells are genetically engineered with pre-defined specificity for melanoma-associated antigens making adoptive cell therapy with melanoma specific T cells possible. In this contribution we will discuss the rationale for adoptive cell therapy of melanoma, evidence for efficacy and current challenges to achieve long-term remission. Upcoming strategies in melanoma stem cell targeting are also discussed.

2. Adoptive therapy with ex vivo amplified TILs can induce regression of melanoma

An effective immune response can control melanoma. This notion is supported by the observation that spontaneous and complete melanoma regressions can occur and that immune compromised patients suffer from a higher frequency of melanoma [17, 18]. The conclusion is moreover sustained by the clinical observation that treatment with high dose IL-2 produces an objective response even in late stage melanoma, some patients with long-term complete response for years [11, 19]. Although about 16%, the response rate is remarkable compared to the low and short-lived response rates of classical therapeutic regimens.

First described in 1969 [20], melanoma is infiltrated by T cells of both effector and helper cell origin which can be expanded to high numbers ex vivo in the presence of IL-2. Pioneered by the NCI-Surgery Branch, such tumor infiltrating T cells (TILs) were selected for melanoma reactivity by incubation on feeder cells expressing melanoma-associated antigens [21] and re-administered in substantial numbers together with high dose IL-2 to the patient (Figure 1). Initial trials produced an objective response rate of 11/20 patients [22] which is remarkable since TILs are obviously capable to fight melanoma even in late stage patients who experienced multiple lines of therapy. Responses, however, were of short duration and TILs did not persist for longer period in the peripheral blood after administration. Subsequent trials identified that the key to successful TIL therapy was the number of TILs administered to the patient, the activity of those cells against melanoma and the rapidity of T cell amplification ex vivo [23, 24]. During the subsequent years the initial protocols were optimized with respect to these and other issues and adopted according to GMP standards [25]; a number of trials are currently open in various centers (Table 1).

Target antigen Adoptively transferred T cells and additional treatment NCT ID Center
TILs, IL-2 in variable dosesNCT00001832NIH
TILs vs. lymphokine-activated killer (LAK) cellsNCT00002535StLMC
TILs, high dose IL-2NCT00096382NIH
TILs, low dose IL-2 s.c.NCT00200577NUH
TILs with vs. without IL-2NCT00314106NIH
TILs, high dose IL-2 with or without dendritic cell immunizationNCT00338377MDACC
TILs, high dose IL-2NCT00604136HMC
TILs, high dose IL-2NCT00863330AHC
TILs, high dose IL-2NCT00937625HUH
TILs, high dose IL-2NCT01005745MOFFITT
TILs, low dose IL-2 and intra-tumoral injection of IFN-γ producing adenovirusNCT01082887NUH
TILs, high dose IL-2NCT01659151MOFFITT
TILs, high dose IL-2NCT01701674MOFFITT
TILs, high dose IL-2NCT01807182FHCRC
"re-stimulated"(autologous DCs &anti-CD3 antibody) TILs, low dose IL-2NCT01883297UHN
TILs, low dose IL-2NCT01883323UHN
TILs, dendritic cell vaccination with NY-ESO-1NCT01946373KUH
TILs, high vs. low dose IL-2NCT01995344CHNHSFT
4-1BB selected TILsNCT02111863NIH
“young“ TILs, high dose IL-2NCT00287131SMC
“young“ TILs with or without CD4+ T cell depletion, high dose IL-2 NCT00513604NIH
“young“ CD8+ TILs, high dose IL-2NCT01118091NIH
“young“ TILs, high dose IL-2NCT01319565NIH
“young“ TILs, high dose IL-2NCT01369875NIH
“young“ TILs, IL-15NCT01369888NIH
“young“ TILsNCT01468818NIH
“young“ TILs, high dose IL-2, BRAF kinase inhibitorNCT01585415NIH
“young“ TILs, with or without high dose IL-2NCT01814046NIH
“young“ TILs, anti-CTLA-4 antibodyNCT01988077SMC
“young“ TILs, high dose IL-2 with standard vs. low dose chemotherapyNCT01993719NIH

Table 1.

Adoptive cell therapy with tumor infiltrating lymphocytes (TILs) in patients with melanoma

[i] - Aurora Health Care; CHNHSFT, Christie Hospital NHS Foundation Trust; FHCRC, Fred Hutchinson Cancer Research Center; HMC, Hadassah Medical Center; HUH, Herlev University Hospital (Copenhagen); KUH, Karolinska University Hospital; MDACC, M.D. Anderson Cancer Center; MOFFITT, H. Lee Moffitt Cancer Center and Research Institute; MUH, Mie University Hospital; NIH, National Institutes of Health; NUH, Nantes University Hospital; SMC, Sheba Medical Center; StLMC, St. Luke's Medical Center; UC, University of California; UHN, University Health Network (Toronto)

Persistence of administered TILs in circulation was substantially improved by depletion of the lymphoid compartment of the patient prior to adoptive cell therapy [26-28]. Such pre-conditioning by non-myeloablative chemotherapy had the effect that cytokines sustaining lymphocyte amplification including IL-7 and IL-15 were present in augmented levels ("cytokine sink"). Moreover, space for transferred lymphocytes was created and suppressor cells including regulatory T cells were depleted which additionally helped to improve engraftment of adoptively transferred T cells.

There are still some issues to be addressed, for instance whether clinically most potent TILs can be defined by phenotype and whether these cells can be selectively expanded. There is a common sense that for therapeutic efficacy in the long-term the functional activity of T cells needs to be preserved without signs of exhaustion which is particularly crucial when T cells experienced extensive amplification ex vivo. In the further development of the procedure, TILs were only selected with respect to their proliferative capacities which is independent of their antigen specificity and represents a furthermore simplification of the standard protocol (Figure 1) [29-31]. Those so-called “young TILs” after short-term ex vivo expansions passed through fewer cell division cycles prior to infusion and are thereby in a maturation stage less prone to terminal differentiation and senescence [32]. Those protocols do not further select TILs for their melanoma reactivity based on the observation that infusion of ex vivo activated, IFN-γ+TILs produced no superior therapeutic efficacy compared to non-responding TILs [16]. These modifications in the protocol resulted in improved persistence of young TILs [33] and about 50% response rates [27, 29, 34], so far in non-randomized trials (Table 1). A series of recent clinical trials with TILs following different lympho-conditioning regimes resulted in objective responses in 56% and complete responses in 22% of patients at the Surgery Branch [35]. Current TIL trials at various centers reproduced objective response rates of 40-50% in melanoma patients, a significant portion of patients free of disease 3-5 years after treatment [36, 37]. Of note, TILs can have anti-tumor activity also towards brain metastases as shown in a NCI trial with 7/17 complete and 6/17 partial remissions [38] sustaining the hope that adoptive cell therapy may be effective towards metastases which are otherwise not accessible.


Figure 1.

T cells used in adoptive cell therapy of melanoma. (A) Tumor infiltrating T cells (TILs) are isolated from melanoma biopsies, selected for reactivity towards melanoma cells, amplified in the presence of IL-2 to clinically relevant numbers and infused to the patient. Alternatively, TILs are expanded without prior selection for melanoma reactivity using a short-term amplification protocol ("young TILs"). (B) T cells from the peripheral blood of melanoma patients are genetically modified by retro- or lentivirus transduction to express a recombinant T cell receptor (TCR) or a chimeric antigen receptor (CAR), specific for a melanoma associated antigen, amplified and administered to the patient.

While most trials apply non-separated TILs, administration of isolated CD8+T cell clones with specificity for Melan-A and gp100 mediated only moderate benefit, required IL-2 and did not persist for longer times [39]. Those CD8+T cells which persisted long-term acquired a phenotype of central memory-type T cells in vivo [40]. It is therefore assumed that CD8+TILs require help of CD4+cells for prolonged persistence making application of non-separated T cell populations more suitable.

Not only the stage of maturation but also the recruitment of T cells through chemokine gradients is crucial for therapeutic success. A recent prospective-retrospective hypothesis-driven analysis revealed that coordinate over-expression of CXCL9, CXCL10, CXCL11, CCL5 in melanoma is associated with responsiveness to treatment after TIL therapy [41].

Melanoma-reactive T cells need to persist in circulation to ensure therapeutic success of TIL therapy [42, 43]. This is reflected by the median survival of patients treated with Melan-A specific TILs of 53.5 months compared to 3.5 months for patients who received TILs of unknown specificity [44]. Some trials are initiated using melanoma specific patient's T cells from the peripheral blood for adoptive cell therapy of melanoma (Table 2). MART-1 or gp100 specific T cell clones isolated and amplified ex vivo produced a 50% response rate [45], however, technical difficulties limit a broad application of such specific T cells since melanoma reactive T cells in the peripheral blood of melanoma patients are extremely rare.

Target antigen Adoptively transferred T cells NCT ID Center
MAGE-1 or MAGE-3melanoma specific CD8+ T cellsNCT00045149FHCRC
Tyrosinasetyrosinase specific CD8+ T cellsNCT00002786FHCRC
MART-1MART-1 specific CD8+ T cells with or without high dose IL-2NCT01495572NIH
MART-1MART-1 specific CD8+ T cells NCT00512889DFCI
MART-1MART-1 specific T cellsNCT00720031NUH
MART-1MART-1 specific CD8+ T cellsNCT00324623CHUV
MART-1MART-1 specific TILs, high dose IL-2NCT00924001NIH
MART-1MART-1 specific CD8+ T cells, low dose IL-2NCT01106235FHCRC
NY-ESO-1NY-ESO-1 specific CD8+ T cells, low dose IL-2, anti-CTLA-4 antibodyNCT00871481FHCRC

Table 2.

Adoptive cell therapy with autologous, antigen specific T cells in patients with melanoma

[i] - CHUV, Centre Hospitalier Universitaire Vaudois; DFCI, Dana-Farber Cancer Institute; FHCRC, Fred Hutchinson Cancer Research Center; NIH, National Institutes of Health; NUH, Nantes University Hospital

3. T cells with engineered anti-melanoma specificity

The success of melanoma antigen specific T cells from peripheral blood strengthened efforts to obtain melanoma-specific T cell clones by genetic engineering of patient's T cells from the peripheral blood. In particular, the molecular cloning of the TCR from melanoma-reactive T cells enabled the engraftment of melanoma specificity to any T cell (Figure 1) [46-49]. A TCR with specificity for gp100 was cloned from melanoma reactive TILs and transferred by retrovirus-mediated gene transfer into blood T cells which thus obtained redirected specificity for gp100+cells in addition to their parental specificity. TCR engineered T cells recognized gp100+melanoma cells, secreted pro-inflammatory cytokines including IFN-γ and lysed gp100+melanoma cells [50, 51]. By the same strategy, blood T cells were modified with the TCR specific for other melanoma associated antigens (Table 3). Using T cells modified with a gp100 specific TCR objective response was induced in 19% of patients, most responses were persistent [49]. Melanoma regression was also obtained in 5/11 melanoma patients after transfer of T cells modified with a TCR that recognizes NY-ESO-1, a protein encoded by a member of the cancer/germline family of genes [52, 53].

Melanoma regression was obtained in about 30% of patients after cell therapy with MART-1 specific T cells [49, 52, 54-56]. As a side effect, patients suffered from vitiligo and destruction of melanocytes in the eye and ear indicating that T cells with engineered specificity can target rare and healthy cells even with the cognate antigen at low levels. In a recent trial, patients were treated with T cells engineered with an anti-MAGE-A3 TCR [57]. While 5/9 patients experienced melanoma regression, three of them had mental status changes and two lapsed into coma and died. Histology revealed necrotizing leukoencephalopathy which is likely due to the recognition of previously unknown epitopes of MAGE-A9/A12, the latter expressed in the brain.

Prolonged clinical remission was observed when engineered T cells persisted in the circulation for longer times; TCR modified T cells were recorded in the blood for more than a year after initiation of treatment [56, 58]. Moreover, TCR engineered T cells were capable to penetrate the blood-brain barrier and to induce regression of brain metastases [57] giving hope that patients with metastases at otherwise incurable sites may benefit from adoptive cell therapy. However, tumor cells may become invisible to TCR modified T cells due to repression of the MHC complex [60], β2 microglobulin mutation [61], and deficiencies in the antigen processing machinery [60, 62], all of them resulting in diminished antigen presentation and less TCR-mediated T cell activation.

Engineering T cells with a recombinant TCR may produce a safety hazard when the transgenic αβ TCR forms hetero-dimers with the respective α and β TCR chains of the endogenous TCR. Such mis-pairing of TCR chains can induce severe auto-reactivity as a result in gain of an unpredictable specificity [63, 64]. The situation was technically solved by different means including replacing the human by the homologous murine constant moieties of the TCR [65] and by inserting additional cysteine bridges [66] to facilitate preferential pairing of the recombinant TCR αβ chains in the presence of the physiologic αβ TCR. These and other technical difficulties of the TCR strategy promoted the development of an artificial “all-in-one" receptor molecule to redirect T cells in an antigen-restricted fashion as summarized below.

Target antigen Adoptively transferred T cells NCT ID Center
IL-12 engineered TILsNCT01236573NIH
IL-2 engineered TILsNCT00062036NIH
CXCR2 and NGFR transduced TILs, high dose IL-2NCT01740557MDACC
TGF-Beta resistant (DNRII) and NGFR transduced TILs, high dose IL-2NCT01955460MDACC
gp-100anti-gp-100 TCR engineered CD8+ cells, anti-gp-100 TCR engineered TILs, high dose IL-2NCT00085462NIH
gp-100anti-gp-100 TCR engineered T cells, high dose IL-2 plus gp-100 vaccinationNCT00610311NIH
gp-100 & MART-1anti-gp-100 TCR & anti-MART-1 TCR engineered T cells high dose IL-2 Peptide ImmunizationNCT00923195NIH
MAGE-A3anti-MAGE-A3/12 TCR engineered T cells, high dose IL-2NCT01273181NIH
MAGE-A3anti-MAGE-A3 TCR engineered T cells, high dose IL-2NCT02153905NIH
MAGE-A3anti-MAGE-A3-DP4 TCR engineered CD4+ cells, high dose IL-2NCT02111850NIH
MAGE-A4anti-MAGE-A4 TCR engineered T cellsNCT02096614MUH
MAGE-A4anti-MAGE-A4 TCR engineered T cellsNCT01694472TMUCIH
MART-1anti-MART-1 TCR engineered T cells, IL-2, peptide immunizationNCT00091104NIH
MART-1anti-MART-1 TCR engineered T cells, IL-2, MART-1 peptide pulsed dendritic cellsNCT00910650UC
MART-1anti-MART-1 TCR engineered T cells, high dose IL-2, peptide immunizationNCT00612222NIH
MART-1anti-MART-1 TCR T cells vs. anti-MART-1 TCR TILs, high dose IL-2NCT00509288NIH
MART-1anti-MART-1 TCR engineered T cells, low dose IL-2, peptide immunizationNCT00706992NIH
NY-ESO-1anti-NY-ESO-1 TCR engineered T cells, high dose IL-2NCT00670748NIH
NY-ESO-1anti-NY ESO-1 mTCR engineered T cells, high dose IL-2NCT01967823NIH
NY-ESO-1anti-NY ESO-1 TCR CD62L+ T cells, high dose IL-2NCT02062359NIH
NY-ESO-1anti-NY-ESO-1 TCR engineered T cellsNCT01350401Adaptimmune
NY-ESO-1anti-NY-ESO-1 TCR engineered T cells, cotransduced with IL-12 cDNANCT01457131NIH
p53anti-p53 TCR engineered T cells, high dose IL-2, p53 peptide pulsed dendritic cellsNCT00704938NIH
p53anti-p53 TCR engineered T cells, high dose IL-2NCT00393029NIH
tyrosinaseanti-tyrosinase(368-376) TCR engineered T cellsNCT01586403LU
GD2 3rd generation anti-GD2 CAR engineered T cellsNCT02107963NIH
VEGFR2anti-VEGFR2 CAR engineered CD8+ T cells, low dose IL-2NCT01218867NIH

Table 3.

Adoptive cell therapy with engineered antigen specific T cells in patients with melanoma

[i] - LU, Loyola University (Chicago); MDACC, M.D. Anderson Cancer Center; MUH, Mie University Hospital; NIH, National Institutes of Health; TMUCIH, Tianjin Medical University Cancer Institute and Hospital; UC, University of California;

4. CAR T cells with engineered specificity for melanoma

In order to link antigen recognition with the downstream signaling machinery of the TCR, Zelig Eshhar (Weizmann Institute of Science) reported a chimeric antigen receptor (CAR) molecule, also named immunoreceptor, which is composed in the extracellular moiety of a single chain fragment of variable region (scFv) antibody for binding and in the intracellular moiety of the CD3ζ endodomain to initiate T cell activation [67]. The coding sequence of such recombinant receptor molecule is transferred by retro- or lentiviral transduction into T cells in vitro (Figure 1) [68]. CAR engineered T cells, also nick-named “T-bodies”, recognize their new target by CAR binding and become activated to secrete pro-inflammatory cytokines, to amplify and to lyse the cognate target cells. Since the binding domain is derived from an antibody the CAR recognizes the target in a MHC-independent fashion which makes major differences to TCR mediated T cell recognition. For instance, the CAR recognizes its target independently of the individual HLA subtype and CAR T cells are not affected by MHC repression and loss of HLA molecules on target cells which frequently occurs during tumor progression. However, recognition by CARs is restricted to target antigens on the cell surface; intracellular antigens like transcription factors are not visible to CAR T cells. Despite that limitation, a nearly infinite variety of targets can be recognized including those which are not classical T cell targets like carbohydrates and gangliosides [69].

Full and lasting T cell activation requires two complementary signals, one provided by the TCR/CD3 and the other by co-receptors the prototype of which is CD28. Prolonged T cell activation, however, requires costimulation and autocrine factors, in particular IL-2 which is only secreted upon TCR and simultaneous CD28 signaling. The lack of appropriate costimulation in the tumor lesion provides the rationale for combining the intracellular CD3ζ with the CD28 signaling domain in one polypeptide chain of a "second generation" CAR (Figure 2). A CAR with combined CD28-CD3ζ signaling domain provides both the primary CD3ζ and the required costimlatory signal when engaging the cognate target. CARs with a costimulatory domain clearly provide clinical benefit and improved T cell persistence compared to CARs with the CD3ζ domain only [70-72]. Other costimulatory moieties, such as 4-1BB (CD137) and OX40 (CD134), also provide full T cell activation when linked to CD3ζ in a CAR; the individual costimulory domains have different impact on T cell effector functions [73]. These and other costimulatory domains were furthermore combined in so-called "3rd generation" CARs which provide advantage for matured effector T cells in terminal differentiation but less in younger stages of T cell development [74]. A number of additional modifications of the CAR design were explored in order to improve T cell persistence and activation and finally the anti-tumor response [75, 76].

While the antibody domain defines the target specificity of the CAR, a plethora of antigens can potentially be used as target for the adoptive cell therapy of melanoma. T cells engineered for targeting melanoma-associated antigens include CARs with specificity for HMW-MAA, also known as MCSP [77, 78], melanotransferrin [79], and the gangliosides GD2 [80] and GD3 [81]. Trials are currently recruiting; to our best knowledge no published data are so far available.

During the last years spectacular efficacy was achieved with CAR T cells in phase I trials for the treatment of lymphoma/leukemia [82, 83]. Clinical response and prolonged T cell activation was accompanied by a "cytokine storm", which occurred even weeks after initial T cell administration; the side effect can clinically be managed by treatment with a neutralizing anti-IL-6 antibody without affecting the anti-tumor efficacy.

The enthusiasm in CAR T cell therapy, however, was dampened by reports on serious adverse events and fatalities after CAR T cell administration [84, 85]. Targeting ErbB2 produced respiratory failure which is thought to be due to low levels of antigen on a number of healthy cells which are sufficient to trigger "on-target-off-organ" T cell activation [86]. This and other serious adverse events emphasize a careful evaluation of potential targets and the necessity for T cell dose escalation studies to balance anti-tumor efficacy and auto-immunity [75, 87, 88].


Figure 2.

T cells with engineered specificity. T cells physiologically recognize their target by the T cell receptor (TCR) complex which is composed of the TCR α and β chain for recognition and the CD3 chains for signaling. The variable regions of each TCR chain (Vα and Vβ) together bind to the MHC presented antigen, Cα and Cβ represent the constant domains. T cells can be genetically engineered with defined specificity by expression of recombinant TCR αβ chains of known specificity. In contrast to the TCR, the chimeric antigen receptor (CAR) is one polypeptide chain composed of a single chain fragment of variable region (scFv) antibody for antigen recognition, the extracellular spacer domain, a trans-membrane domain and the intracellular CD3ζ ("first generation" CAR), the CD28-CD3ζ ("second generation" CAR), or the CD28-OX40-CD3ζ ("third generation" CAR) signaling chain.

5. "Melanoma stem cells": Target cells to achieve long-term remission?

Despite the tremendous cellular and phenotypic heterogeneity in tumor lesions, cancer is thought to be initiated and maintained by so-called cancer stem cells (CSCs). Such pluripotent stem cells are of low abundance, induce tumors upon transplantation under limiting dilution conditions, resist radiation and chemotherapy, and drive self-renewal and a-symmetric differentiation into a variety of cell types. Residual CSCs are thought to initiate cancer relapse even after years of “dormancy”, which can be more than a decade after surgical treatment of the primary lesion [89]. While the concept of the hierarchical organization in driving tumor progression was initially drawn upon deciphering hematological malignancies, basically the same organization was subsequently reported for other solid cancers including mammary, prostate, pancreatic, colon carcinoma and glioma [90-94].

Transplantation of melanoma cell subsets into recipient mice under limiting dilution conditions also revealed that a defined subset of cancer cells, and not every cell from the same biopsy, can induce tumors of same histology as the parental tumor [90, 95-97]. One conclusion is that melanoma is organized in a hierarchical manner originating from a particular initiator cell, the cancer stem cell, which gives rise to the described diversity of cells in an established lesion. Melanoma initiating cells were described by various, but not common markers, including the transporter protein ABCB5 [95], CD20 [97], or the nerve growth factor receptor CD271 [98]. While CD271+melanoma cells are present in a frequency of approximately 1/2000 cells [98], transplantation under more rigorous conditions, i.e., ideally of one single melanoma cell, revealed that nearly every fourth randomly taken melanoma cell (1/2-1/15) can induce tumors in the host animal. This observation, however, questioned the validity of the stem cell paradigm for melanoma [99, 100]. Subsequent studies made clear that the potential to induce melanoma is not closely associated with a particular phenotype and that the number of potential CSCs in melanoma may not necessarily be low. If nearly every melanoma cell is capable to re-program to a tumor initiating cell under certain conditions, blocking stem cell properties in melanoma will reduce tumor initiation and growth in a transplantation model finally resulting in melanoma ablation [101].

Once the tumor lesion is established, a minor subset of cancer cells seems to take over to control malignant progression. Evidence for this hypothesis was provided from a pre-clinical model [79] which asked whether all or a defined subset of melanoma cells in an established xeno-transplanted lesion need to be eliminated to cause tumor regression. Such melanoma sustaining cell may be, but not must be identical to melanoma stem cells identified by transplantation assays.

Evidence for a particular targetable melanoma cell subset which sustains tumor progression was provided by the observation that elimination of CD20+melanoma cells by adoptive transfer of CAR T cells completely eradicated xeno-transplanted melanoma. Those human melanoma biopsies contained a subset of CD20+melanoma cells which constituted about 1-2% of melanoma cells and which are present in different histological melanoma types and tumor stages. A caveat is that in approximately 20% of melanoma samples analyzed so far, no CD20+melanoma cells were detected by histological screening; CD20-specific CAR T cells did not induce regression of those transplanted tumor lesions. Interestingly, CD20 re-expression in a random subpopulation of those tumor cells by genetic modification did not render the tumor lesion sensitive for eradication indicating that CD20 expression per se is not sufficient but requires additional capabilities to sustain melanoma progression [79].

There are additionally clinical observations that sustain the notion of CD20+cells in promoting melanoma progression. Firstly, a patient with stage III/IV metastatic, refractory melanoma and 2% CD20+melanoma cells who received intra-lesional injections of the anti-CD20 therapeutic antibody rituximab experienced lasting remission accompanied by a decline of the melanoma serum marker S-100 to physiological levels and a switch of a T helper-2 to a more pro-inflammatory T helper-1 response [102]. Although anecdotic, data provide the first clinical evidence that targeted elimination of CD20+melanoma cells can produce regression of chemotherapy-refractory melanoma. Secondly, in a small pilot trial, stage IV melanoma patients without evidence of disease by way of surgery, chemo-and/or radiation therapy received the anti-CD20 antibody systemically for a 2 year period [103]. Data suggest a benefit of anti-CD20 therapy in overall and recurrence-free survival; a caveat being that the number of patients is still small for definitive conclusions.

Currently, the hierarchical stem cell model in the maintenance of an established melanoma is supported by some experimental evidence [79], whereas a body of information on melanoma initiation by transplantation of single melanoma cells sustains the stochastic model [99, 100], although not confirmed by others [98]. The most determining proof of the stem cell hypothesis, however, will be the successful melanoma elimination by targeting stem cells or stem cell properties. For the development of such therapeutic strategies several aspects need to be taken into account.

First, standard therapy will rapidly de-bulk the tumor lesion and the remaining melanoma stem cells, which are more chemo-and radiation resistant, will drive relapse of the disease. Since those melanoma initiating cells are merely in a "dormant" state and replicate less frequently than the majority of melanoma cells in the same lesion, anti-proliferative strategies by classical chemotherapeutic drugs are unlikely efficient. Transporter systems including ABCB5, which is highly expressed by melanoma stem cells [95], additionally contribute to chemotherapy resistance; the chemotherapy and/or radiation itself may promote expression of those transporter systems and survival of those resistant cells which finally contributes to relapse of the disease.

Second, if clinical progression correlates with the prevalence of CD20+melanoma cells, targeted elimination of those melanoma cells requires to meet the fact that those target cells are a small minority. Targeted elimination, e.g., by CD20 redirected cytotoxic T cells or by CD20-specific therapeutic antibodies like Rituxan™ (rituximab) or Arzerra™ (ofatumumab), will be required to obtain substantial efficacy.

Third, the extraordinary functional and phenotypic plasticity of melanoma cells may make it necessary to have the therapeutic agent in place for a longer time. In their pre-clinical model, Schmidt and colleagues [79] used CAR T cells which persist for long-term acting as an antigen-specific guardian as long as target cells are present. Since repetitive re-stimulation sustains the persistence and amplification of CAR T cells, cellular therapy has a major advantage compared to pharmaceutical drugs with a comparable short half-life. CAR T cells can moreover provide antigen-specific memory with defined specificity [104], potentially contributing to control melanoma in the long-term.

6. Production of engineered T cells for clinical application

Application of adoptive cell therapy to clinical use requires efficient production of cells according to good manufacturing practice (GMP). This particularly applies to patient’s T cells which are ex vivo genetically modified. The vector used for T cell modification is of major relevance with respect to the efficiency and stability in modification. Crucial steps in this process are the stable integration of the genetic vector, the site of integration to avoid insertion mutagenesis, and the resistance of the vector to genetic repression. To date, most clinical trials were performed employing retroviral or lentiviral vectors which fulfill some but not all of these requirements. Recently, other vector systems including RNA modification are alternatively utilized and it is expected that these systems will be explored in parallel in the near future.

The way of stimulating the T cells ex vivo for genetic modification and subsequent amplification is crucial for both the success in transduction and the functional capacities of modified cells. T cells are commonly activated by TCR/CD3 stimulation in addition to IL-2 [105]; most protocols use anti-CD3 and anti-CD28 magnetic beads [83, 106] which can be easily eliminated during the production process. IL-2 is replaced by other cytokines such as IL-7 and IL-15 to obtain a T cell population with a more naive and central memory phenotype [107]. Alternatively, cell lines were engineered, so-called “artificial APCs”, which are modified with the various co-stimulating molecules to mimic the physiological stimulation and to provide the required signals [108]. However, difficulties in adopting those cells to GMP standard prevent their broad application in large scale production processes.

For the production itself, static culture systems in flasks or gas permeable bags are traditionally used. Due to their amplification at low cell densities (0.25-1x106 cells per ml), high culture volumes are required to obtain clinically relevant T cell numbers which is more easily achieved by non-static systems including the WAVE-Bioreactor or the G-Rex100 device [83, 106, 109]. In order to produce engineered T cells for a large number of patients it will be required to manufacture cells in a closed system and to produce multiple batches in parallel in the same clean room facility without the risk of batch contamination.

7. Challenges and promise in the adoptive cell therapy of melanoma

To date, approximately half of the melanoma patients benefit from adoptive cell therapy with TILs. Specifically targeted T cells may further improve the therapeutic response. Despite substantial success, the strategy still has major challenges which need to be addressed in the near future.

Significant numbers of effector T cells have to accumulate in the targeted tumor lesion which is mediated by a network of chemokines. Adoptively transferred T cells use these networks to accumulate at the tumor site; melanoma cells secrete a number of chemokines including CXCL1 to attract lymphocytes. However, early imaging studies revealed that melanoma-specific T cells massively infiltrate the lungs, spleen and liver with only some accumulation at the tumor site before the cells decline to undetectable levels in circulation [110-112]. To improve tumor targeting TILs were engineered with CXCR2, the receptor for melanoma secreted CXCL1, which resulted in improved anti-tumor activity in a mouse model [113]. The strategy is currently being explored in an early phase I trial (Table 3) [113].

Since tumor eradication requires a beneficial T cell-to-target cell ratio, higher numbers of tumor-specific T cells applied per dose likely increase the clinical efficacy. The optimal dose of T cells, however, is still a matter of discussion and requires empiric evaluation. A number of trials, in particular applying TILs, administered up to 1010 cells per dose [27]. Such high doses in turn require extended expansions of T cells ex vivo with the risk of loss of the "young" phenotype and gain of more matured T cells. Highly expanded T cells become hypo-responsive to CD28 costimulation and rapidly enter activation induced cell death, in particular upon IL-2 driven expansion [114]. With respect to more potent effector functions short-term amplification protocols are envisioned for both TILs and engineered T cells. This may be achieved by T cell amplification in the presence of IL-15 and IL-21 and/or by 4-1BB co-stimulation [115].

On the other hand, administration of about 105 engineered T cells induced remarkable therapeutic efficacy in recent trials targeting CD19+ leukemia [83]. Since the T cells substantially amplify in vivo upon antigen encounter, the capacity of cells to amplify under appropriate conditions is more relevant than the applied cell number.

Once targeted in sufficient numbers to the tumor tissue, a major challenge is the tumor selectivity of redirected T cells. While the TCR and the CAR is specific for a particular target, in most cases the targeted antigen is not exclusively expressed by cancer but also by healthy cells, although sometimes at lower levels [116, 117]. MART-1, frequently expressed by the majority of melanoma cells, is also expressed by melanocytes. Targeting such type of antigen frequently produces vitiligo, sometimes also inner ear toxicity with a certain degree of deafness [49]. Since nearly all "tumor-associated antigens" which are frequently used as targets for adoptive cell therapy are self-antigens, strategies are needed to minimize such off-target toxicities. Among these, low-avidity TCRs or CARs or combinatorial antigen recognition by two CARs are currently explored.

Melanoma cells may become invisible to TILs or TCR modified T cells due to down-regulation of their MHC components or due to deficiencies in antigen processing. However, melanoma cells may still be visible to CAR T cells which recognized their target by their antibody-derived binding domain in a MHC independent fashion. On the other hand, TCR T cells are capable to recognize cross-presented antigen, for instance tumor antigen presented by stroma cells, which is invisible to CAR T cells but helps to destroy the tumor lesion in the long-term [118, 119].

Consequently, a TCR-like CAR aims at combining the benefits of TCR and CAR redirected T cells. This is performed by using a single chain antibody with TCR-like specificity for recognizing MHC presented antigen. T cells with such a TCR-like CAR were successfully redirected in a MHC restricted fashion towards NY-ESO-1 and MAGE-A1, respectively [120, 121].

The redirected T cell activation depends on the amount of target antigen and binding affinity. Compared to TILs and TCR modified T cells, CAR T cells bind with extraordinary high affinity by their antibody-derived CAR binding domain. A furthermore increase in affinity by affinity maturation does not necessarily improve CAR redirected T cell activation [120, 122]; CD28 costimulation does not add to the affinity dependent activation threshold, however, prolongs T cell persistence and resistance to apoptosis [123]. Targeting cancer cells also depends on the amount of target antigen in addition to the binding affinity. Low affinity CARs require abundant antigen levels for efficient activation of engineered T cells while high affinity CARs are likewise effective against low antigen levels on target cells. In this context, the selectivity in targeting melanoma cells versus healthy cells needs to be discussed not only with respect to the targeted antigen itself but also to antigen amount and binding affinity.

Amplification and persistence of adoptively transferred cells correlates with clinical outcome in some trials [124]. T cells will persist in detectable numbers as long as targeted antigen is present, however, will contract to undetectable levels and disappear from circulation when no target is furthermore present. To enable survival of CAR T cells in the long-term, Epstein-Barr virus (EBV)-specific T cells were used as effector cells and modified with a tumor-specific CAR. The rationale is that EBV specific T cells are maintained in a sizable population in circulation by recognizing EBV antigens by their physiological TCR. The strategy is sustained by the first clinical observation that EBV-specific T cells engineered with an anti-GD2 CAR showed benefit over non-virus-specific, CAR engineered T cells in the treatment of neuroblastoma (NCT00085930) [124]. Other trials use EBV or CMV specific, autologous T cells engineered with a first or second generation CAR, for instance directed against HER2/neu (ErbB2) (NCT01109095), CD30 (NCT01192464), or CD19 (NCT00709033; NCT01475058; NCT01430390; NCT00840853; NCT01195480).

The T cell subset matters, adoptively transferred CD8+T cell clones poorly persist [125] and need help of CD4+cells. Prolonged T cell anti-tumor response also requires resistance to repression in the tumor tissue. A number of efforts are currently undertaken to counteract tumor associated T cell repression, in particular mediated by Treg cells and checkpoint mediators. In animal models, CD28 costimulation without induction of IL-2 secretion protects a CAR redirected T cell response from Treg cell repression [126]. On the other hand, repetitive T cell stimulation upregulates CTLA-4 which acts as negative regulator to return the T cell to a resting stage. Administration of a CTLA-4 blocker, e.g., ipilimumab antibody, may prolong the anti-tumor activation of transferred T cells, although it is not locally restricted and will likewise affect all T cells [127, 128]. Expression profiling of TCR-engineered T cells demonstrates overexpression of multiple inhibitory receptors in persisting lymphocytes, including PD-1 and CD160, the latter associated with decreased reactivity of TCR T cells in a ligand independent manner [129]. Essentially the same was observed for CAR T cells [130]. These analyses point to a multi-factorial T cell repression in the tumor tissue; there is more than one uni-directional strategy needed to sustain the T cell anti-tumor response in the long-term.

A major hurdle of specific immunotherapy in general is the tremendous heterogeneity of cancer cells within the same lesion. Low or loss of target antigen expression negatively affects the long-term therapeutic efficacy of an antigen-redirected approach. This is supported by several reports which document a relapse of antigen-loss tumor metastases after adoptive therapy with melanoma-reactive T cell clones [39,131, 132]. A solution may be the use of polyclonal T cells with specificities for various melanoma antigens or T cells modified with different CARs recognizing different antigens; however, target-negative tumor cells will not be recognized. On the other hand, pro-inflammatory cytokines secreted by redirected T cells upon activation can attract a second wave of innate immune cells which in turn may eradiate the antigen-negative tumor cells. At least in an animal model, antigen-negative melanoma cells are indeed eliminated when co-inoculated with antibody-targeted cytokines [133]. T cells engineered with induced expression of transgenic IL-12 can attract innate immune cells including macrophages into the tumor tissue which eliminate antigen-negative tumor cells in the same lesion, at least in an immune competent animal model [134]. Such "TRUCK" cells ("T cells redirected for unrestricted cytokine killing") may pave a novel way to deliver transgenic cell products to pre-defined, target lesions.

Combination of adoptive cell therapy with pathway inhibitors may improve the efficacy in melanoma cell elimination, in particular in disseminated stages of the disease. Metastatic melanoma patients with the B-raf activating mutation V600E benefit from a small molecule drug, PLX4032 or vemurafenib, which inhibits the mitogen-activated protein kinase (MAPK) pathway. Treatment with vemurafenib is accompanied by increased T cell infiltrations in the melanoma lesions [135, 136] which may contribute to the therapeutic effect and may be improved by co-administration of melanoma-specific T cells.

While adoptive cell therapy is mostly performed with modified or non-modified T cells, other cells like monocytes, macrophages as well as NK cells can also be redirected by CARs in an antigen-specific fashion [137-141, 144]. In contrast to T cells, NK cells can be rapidly activated and exhibit high cytotoxic potential and continuously growing NK cell lines such as NK-92 can be used for adoptive cancer immunotherapy [142]. CD3ζ chain CARs trigger cytolytic activities of NK cells which has been shown for CARs with various specificities [138, 141, 143-147]. Similar to T cells, the anti-tumor activity was improved by adding 4-1BB or 2B4 (CD244) costimulatory domains [148, 149]. Since NK cells cannot provide IL-2 or IL-15 required for amplification, CAR modified NK cells were additionally engineered to release IL-15 which sustains NK cell expansion and CAR-mediated cytotoxicity in the absence of IL-2 [150]. Despite these and other advances during the last years, experience with CAR engineered primary NK cells in clinical trials is still limited; whether redirected cells of the innate immune system are more advantageous in melanoma elimination than modified T cells has moreover to be explored in clinical trials.


Work in the author's laboratory was supported by the Deutsche Forschungsgemeinschaft, Bonn, Deutsche Krebshilfe, Bonn, Else Kröner-Fresenius-Stiftung, Bad Homburg v.d.H., Wilhelm Sander-Stiftung, München, the European Union (European Regional Development Fund-Investing in your future), the German federal state North Rhine-Westphalia (NRW), and the Fortune program of the Medical Faculty of the University of Cologne.


1 - Garbe C, Peris K, Hauschild A, Saiag P, Middleton M, Spatz A, Grob J-J, Malvehy J, Newton-Bishop J, Stratigos A, Pehamberger H, Eggermont A. Diagnosis and treatment of melanoma: European consensus-based interdisciplinary guideline. European Journal of Cancer 2010;46(2) 270-283.
2 - Denninghoff VC, Kahn AG, Falco J, Curutchet HP, Elsner B. Sentinel lymph node: detection of micrometastases of melanoma in a molecular study. Molecular Diagnosis 2004;8(4) 253-258.
3 - Bedikian AY, Wei C, Detry M, Kim KB, Papadopoulos NE, Hwu WJ, Homsi J, Davies M, McIntyre S, Hwu P. Predictive Factors for the Development of Brain Metastasis in Advanced Unresectable Metastatic Melanoma. American Journal of Clinical Oncology 2010;34(6) 603-610.
4 - Chapman PB, Hauschild A, Robert C, Haanen JB, Ascierto P, Larkin J, Dummer R, Garbe C, Testori A, Maio M, Hogg D, Lorigan P, Lebbe C, Jouary T, Schadendorf D, Ribas A, O'Day SJ, Sosman JA, Kirkwood JM, Eggermont AM, Dreno B, Nolop K, Li J, Nelson B, Hou J, Lee RJ, Flaherty KT. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. The New England Journal of Medicine 2011;364 (26) 2507-2516.
5 - Carter RD, Krementz ET, Hill GJ 2nd, Metter GE, Fletcher WS, Golomb FM, Grage TB, Minton JP, Sparks FC. DTIC (nsc-45388) and combination therapy for melanoma. I. Studies with DTIC, BCNU (NSC-409962), CCNU (NSC-79037), vincristine (NSC-67574), and hydroxyurea (NSC-32065). Cancer Treat Rep. 1976;60(5) 601-609.
6 - Flaherty KT, Puzanov I, Kim KB, Ribas A, McArthur GA, Sosman JA, O'Dwyer PJ, Lee RJ, Grippo JF, Nolop K, Chapman PB. Inhibition of mutated, activated BRAF in metastatic melanoma. The New England Journal of Medicine 2010;363(9) 809-819.
7 - Leiter U, Eigentler TK, Forschner A, Pflugfelder A, Weide B, Held L, Meier F, Garbe C. Excision guidelines and follow-up strategies in cutaneous melanoma: Facts and controversies. Clinics in Dermatology 2010;28(3) 311-315.
8 - Bradbury PA, Middleton MR. DNA repair pathways in drug resistance in melanoma. Anti-cancer Drugs 2004;15(5) 421-426.
9 - Pak BJ, Chu W, Lu SJ, Kerbel RS, Ben-David Y. Lineage-specific mechanism of drug and radiation resistance in melanoma mediated by tyrosinase-related protein 2. Cancer Metastasis Reviews 2001;20(1-2) 27-32.
10 - Pak BJ, Lee J, Thai BL, Fuchs SY, Shaked Y, Ronai Z, Kerbel RS, Ben-David Y. Radiation resistance of human melanoma analysed by retroviral insertional mutagenesis reveals a possible role for dopachrome tautomerase. Oncogene 2004;23(1) 30-38.
11 - Atkins MB, Lotze MT, Dutcher JP, Fisher RI, Weiss G, Margolin K, Abrams J, Sznol M, Parkinson D, Hawkins M, Paradise C, Kunkel L, Rosenberg SA. High-dose recombinant interleukin 2 therapy for patients with metastatic melanoma: analysis of 270 patients treated between 1985 and 1993. Journal of Clinical Oncology 1999;17(7) 2105-2116.
12 - Hodi FS, O'Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen JB, Gonzalez R, Robert C, Schadendorf D, Hassel JC, Akerley W, van den Eertwegh AJ, Lutzky J, Lorigan P, Vaubel JM, Linette GP, Hogg D, Ottensmeier CH, Lebbé C, Peschel C, Quirt I, Clark JI, Wolchok JD, Weber JS, Tian J, Yellin MJ, Nichol GM, Hoos A, Urba WJ. Improved survival with ipilimumab in patients with metastatic melanoma. The New England Journal of Medicine 2010;363(8) 711-723.
13 - Kirkwood JM, Ibrahim JG, Sondak VK, Richards J, Flaherty LE, Ernstoff MS, Smith TJ, Rao U, Steele M, Blum RH. High-and low-dose interferon alfa-2b in high-risk melanoma: first analysis of intergroup trial E1690/S9111/C9190. Journal of Clinical Oncology 2000;18(12) 2444-2458.
14 - Kirkwood JM, Strawderman MH, Ernstoff MS, Smith TJ, Borden EC, Blum RH. Interferon alfa-2b adjuvant therapy of high-risk resected cutaneous melanoma: the Eastern Cooperative Oncology Group Trial EST 1684. Journal of Clinical Oncology 1996;14(1) 7-17.
15 - Galluzzi L, Vacchelli E, Eggermont A, Fridman WH, Galon J, Sautès-Fridman C, Tartour E, Zitvogel L, Kroemer G. Trial Watch: Adoptive cell transfer immunotherapy. Oncoimmunology 2012;1(3) 306-315.
16 - Bernatchez C, Radvanyi LG, Hwu P. Advances in the treatment of metastatic melanoma: adoptive T-cell therapy. Seminars in Oncology 2012;39(2) 215-226.
17 - Grulich AE, van Leeuwen MT, Falster MO, Vajdic CM. Incidence of cancers in people with HIV/AIDS compared with immunosuppressed transplant recipients: a meta-analysis. Lancet 2007;370(9581) 59-67.
18 - Nathanson. Spontaneous regression of malignant melanoma: a review of the literature on incidence, clinical features, and possible mechanisms. National Cancer Institute Monograph 1976;44 67-76.
19 - Rosenberg SA, Yang JC, Topalian SL, Schwartzentruber DJ, Weber JS, Parkinson DR, Seipp CA, Einhorn JH, White DE. Treatment of 283 consecutive patients with metastatic melanoma or renal cell cancer using high-dose bolus interleukin 2. The journal of the American Medical Association 1994;271(12) 907-913.
20 - Clark WH Jr, From L, Bernardino EA, Mihm MC. The histogenesis and biologic behavior of primary human malignant melanomas of the skin. Cancer Research 1969;29(3) 705-727.
21 - Vignard V, Lemercier B, Lim A, Pandolfino MC, Guilloux Y, Khammari A, Rabu C, Echasserieau K, Lang F, Gougeon ML, Dreno B, Jotereau F, Labarriere N. Adoptive transfer of tumor-reactive Melan-A-specific CTL clones in melanoma patients is followed by increased frequencies of additional Melan-A-specific T cells. Journal of Immunology 2005;175(7) 4797-4805.
22 - Rosenberg SA, Packard BS, Aebersold PM, Solomon D, Topalian SL, Toy ST, Simon P, Lotze MT, Yang JC, Seipp CA, et al. Use of tumor-infiltrating lymphocytes and interleukin-2 in the immunotherapy of patients with metastatic melanoma. N. Engl. J. Med. 1988;319(25), 1676–1680.
23 - Clemente CG, Mihm MC Jr, Bufalino R, Zurrida S, Collini P, Cascinelli N. Prognostic value of tumor infiltrating lymphocytes in the vertical growth phase of primary cutaneous melanoma. Cancer 1996;77(7) 1303-1310.
24 - Burton AL, Roach BA, Mays MP, Chen AF, Ginter BA, Vierling AM, Scoggins CR, Martin RC, Stromberg AJ, Hagendoorn L, McMasters KM. Prognostic significance of tumor infiltrating lymphocytes in melanoma. The American Surgeon 2011;77(2) 188-192.
25 - Hinrichs CS, Rosenberg SA. Exploiting the curative potential of adoptive T-cell therapy for cancer. Immunological Reviews Adoptive Immunotherapy for Cancer 2014; 257(1) 56–71.
26 - Dudley ME, Wunderlich JR, Robbins PF, Yang JC, Hwu P, Schwartzentruber DJ, Topalian SL, Sherry R, Restifo NP, Hubicki AM, Robinson MR, Raffeld M, Duray P, Seipp CA, Rogers-Freezer L, Morton KE, Mavroukakis SA, White DE, Rosenberg SA. Cancer regression and autoimmunity in patients after clonal repopulation with anti-tumor lymphocytes. Science 2002;298(5594) 850–854.
27 - Dudley ME, Wunderlich JR, Yang JC, Sherry RM, Topalian SL, Restifo NP, Royal RE, Kammula U, White DE, Mavroukakis SA, Rogers LJ, Gracia GJ, Jones SA, Mangiameli DP, Pelletier MM, Gea-Banacloche J, Robinson MR, Berman DM, Filie AC, Abati A, Rosenberg SA. Adoptive cell transfer therapy following non-myeloablative but lymphodepleting chemotherapy for the treatment of patients with refractory metastatic melanoma. Journal of Clinical Oncology 2005;23(10) 2346-2357.
28 - Dudley ME, Yang JC, Sherry R, Hughes MS, Royal R, Kammula U, Robbins PF, Huang J, Citrin DE, Leitman SF, Wunderlich J, Restifo NP, Thomasian A, Downey SG, Smith FO, Klapper J, Morton K, Laurencot C, White DE, Rosenberg SA. Adoptive cell therapy for patients with metastatic melanoma: evaluation of intensive myeloablative chemoradiation preparative regimens. Journal of Clinical Oncology 2008;26(32) 5233-5239.
29 - Besser MJ, Shapira-Frommer R, Treves AJ, Zippel D, Itzhaki O, Hershkovitz L, Levy D, Kubi A, Hovav E, Chermoshniuk N, Shalmon B, Hardan I, Catane R, Markel G, Apter S, Ben-Nun A, Kuchuk I, Shimoni A, Nagler A, Schachter J. Clinical responses in a phase II study using adoptive transfer of short-term cultured tumor infiltration lymphocytes in metastatic melanoma patients. Clinical Cancer Research 2010;16(9) 2646-2655.
30 - Tran KQ, Zhou J, Durflinger KH, Langhan MM, Shelton TE, Wunderlich JR, Robbins PF, Rosenberg SA, Dudley ME. Minimally cultured tumor-infiltrating lymphocytes display optimal characteristics for adoptive cell therapy. J. Immunother 2008;31(8) 742–751.
31 - Dudley ME, Gross CA, Langhan MM, Garcia MR, Sherry RM, Yang JC, Phan GQ, Kammula US, Hughes MS, Citrin DE, Restifo NP, Wunderlich JR, Prieto PA, Hong JJ, Langan RC, Zlott DA, Morton KE, White DE, Laurencot CM, Rosenberg SA. CD8+enriched "young" tumor infiltrating lymphocytes can mediate regression of metastatic melanoma. Clinical Cancer Research 2010;16(24) 6122-6131.
32 - Itzhaki O, Hovav E, Ziporen Y, Levy D, Kubi A, Zikich D, Hershkovitz L, Treves AJ, Shalmon B, Zippel D, Markel G, Shapira-Frommer R, Schachter J, Besser MJ. Establishment and large-scale expansion of minimally cultured "young" tumor infiltrating lymphocytes for adoptive transfer therapy. Journal of Immunology 2011;34(2) 212-220.
33 - Shen X, Zhou J, Hathcock KS, Robbins P, Powell DJ Jr, Rosenberg SA, Hodes RJ. Persistence of tumor infiltrating lymphocytes in adoptive immunotherapy correlates with telomere length. Journal of Immunology 2007;30(1) 123-129.
34 - Besser MJ, Shapira-Frommer R, Treves AJ, Zippel D, Itzhaki O, Schallmach E, Kubi A, Shalmon B, Hardan I, Catane R, Segal E, Markel G, Apter S, Nun AB, Kuchuk I, Shimoni A, Nagler A, Schachter J. Minimally cultured or selected autologous tumor-infiltrating lymphocytes after a lympho-depleting chemotherapy regimen in metastatic melanoma patients. Journal of Immunotherapy 2009;32(4) 415-423.
35 - Rosenberg SA, Yang JC, Sherry RM, Kammula US, Hughes MS, Phan GQ, Citrin DE, Restifo NP, Robbins PF, Wunderlich JR, Morton KE, Laurencot CM, Steinberg SM, White DE, Dudley ME. Durable complete responses in heavily pretreated patients with metastatic melanoma using T-cell transfer immunotherapy. Clin Cancer Res 2011;17(13) 4550–4557.
36 - Radvanyi LG, Bernatchez C, Zhang M, Fox PS, Miller P, Chacon J, Wu R, Lizee G, Mahoney S, Alvarado G, Glass M, Johnson VE, McMannis JD, Shpall E, Prieto V, Papadopoulos N, Kim K, Homsi J, Bedikian A, Hwu WJ, Patel S, Ross MI, Lee JE, Gershenwald JE, Lucci A, Royal R, Cormier JN, Davies MA, Mansaray R, Fulbright OJ, Toth C, Ramachandran R, Wardell S, Gonzalez A, Hwu P. Specific lymphocyte subsets predict response to adoptive cell therapy using expanded autologous tumor-infiltrating lymphocytes in metastatic melanoma patients. Clin Cancer Res 2012;18 (24) 6758–6770.
37 - Itzhaki O, Hovav E, Ziporen Y, Levy D, Kubi A, Zikich D, Hershkovitz L, Treves AJ, Shalmon B, Zippel D, Markel G, Shapira-Frommer R, Schachter J, Besser MJ. Establishment and large-scale expansion of minimally cultured “young” tumor infiltrating lymphocytes for adoptive transfer therapy. J Immunother 2011;34(2) 212–220.
38 - Hong JJ, Rosenberg SA, Dudley ME, Yang JC, White DE, Butman JA, Sherry RM. Successful treatment of melanoma brain metastases with adoptive cell therapy. Clin Cancer Res 2006;16(19), 4892–4898.
39 - Yee C, Thompson JA, Byrd D, Riddell SR, Roche P, Celis E, Greenberg PD. Adoptive T cell therapy using antigen-specific CD8+T cell clones for the treatment of patients with metastatic melanoma: in vivo persistence, migration, and antitumor effect of transferred T cells. Proc Natl Acad Sci USA (2002);99(25) 16168–16173.
40 - Inozume T, Hanada K, Wang QJ, Ahmadzadeh M, Wunderlich JR, Rosenberg SA, Yang JC. (2010) Selection of CD8+PD-1+lymphocytes in fresh human melanomas enriches for tumor-reactive T cells. J Immunother(2010); 33(9) 956–964.
41 - Bedognetti D, Spivey TL, Zhao Y, Uccellini L, Tomei S, Dudley ME, Ascierto ML, De Giorgi V, Liu Q, Delogu LG, Sommariva M, Sertoli MR, Simon R, Wang E, Rosenberg SA, Marincola FM. CXCR3/CCR5 pathways in metastatic melanoma patients treated with adoptive therapy and interleukin-2. British Journal of Cancer 2013;109(9) 2412–2423.
42 - Vignard V, Lemercier B, Lim A, Pandolfino MC, Guilloux Y, Khammari A, Rabu C, Echasserieau K, Lang F, Gougeon ML, Dreno B, Jotereau F, Labarriere N. Adoptive transfer of tumor-reactive Melan-A-specific CTL clones in melanoma patients is followed by increased frequencies of additional Melan-A-specific T cells. Journal of Immunology 2005;175(7) 4797-4805.
43 - Kawakami Y, Eliyahu S, Jennings C, Sakaguchi K, Kang X, Southwood S, Robbins PF, Sette A, Appella E, Rosenberg SA. Recognition of multiple epitopes in the human melanoma antigen gp100 by tumor-infiltrating T lymphocytes associated with in vivo tumor regression. Journal of Immunology 1995;154(8) 3961-39618.
44 - Benlalam H, Vignard V, Khammari A, Bonnin A, Godet Y, Pandolfino MC, Jotereau F, Dreno B, Labarrière N. Infusion of Melan-A/Mart-1 specific tumor-infiltrating lymphocytes enhanced relapse-free survival of melanoma patients. Cancer Immunology Immunotherapy 2007;56(4) 515-526.
45 - Khammari A, Labarrière N, Vignard V, Nguyen JM, Pandolfino MC, Knol AC, Quéreux G, Saiagh S, Brocard A, Jotereau F, Dreno B. Treatment of metastatic melanoma with autologous Melan-A/MART-1-specific cytotoxic T lymphocyte clones. The Journal of Investigative Dermatology 2009;129(12) 2835-2842.
46 - Johnson LA, Heemskerk B, Powell DJ Jr, Cohen CJ, Morgan RA, Dudley ME, Robbins PF, Rosenberg SA. Gene transfer of tumor-reactive TCR confers both high avidity and tumor reactivity to nonreactive peripheral blood mononuclear cells and tumor-infiltrating lymphocytes. Journal of Immunology 2006;177(9) 6548-6559.
47 - Zhao Y, Zheng Z, Khong HT, Rosenberg SA, Morgan RA. Transduction of an HLA-DP4-restricted NY-ESO-1-specific TCR into primary human CD4+lymphocytes. Journal of Immunology 2006;29(4) 398-406.
48 - Frankel TL, Burns WR, Peng PD, Yu Z, Chinnasamy D, Wargo JA, Zheng Z, Restifo NP, Rosenberg SA, Morgan RA. Both CD4 and CD8 T cells mediate equally effective in vivo tumor treatment when engineered with a highly avid TCR targeting tyrosinase. Journal of Immunology 2010;184(11) 5988-5998.
49 - Johnson LA, Morgan RA, Dudley ME, Cassard L, Yang JC, Hughes MS, Kammula US, Royal RE, Sherry RM, Wunderlich JR, Lee CC, Restifo NP, Schwarz SL, Cogdill AP, Bishop RJ, Kim H, Brewer CC, Rudy SF, VanWaes C, Davis JL, Mathur A, Ripley RT, Nathan DA, Laurencot CM, Rosenberg SA. Gene therapy with human and mouse T-cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen. Blood 2009;114(3) 535-546.
50 - Schaft N, Willemsen RA, de Vries J, Lankiewicz B, Essers BW, Gratama JW, Figdor CG, Bolhuis RL, Debets R, Adema GJ. Peptide fine specificity of anti-glycoprotein 100 CTL is preserved following transfer of engineered TCR alpha beta genes into primary human T lymphocytes. Journal of Immunology 2003;170(4) 2186-2194.
51 - Morgan RA, Dudley ME, Yu YY, Zheng Z, Robbins PF, Theoret MR, Wunderlich JR, Hughes MS, Restifo NP, Rosenberg SA. 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. Journal of Immunology 2003;171(6) 3287-3295.
52 - Robbins PF, Morgan RA, Feldman SA, Yang JC, Sherry RM, Dudley ME, Wunderlich JR, Nahvi AV, Helman LJ, Mackall CL, Kammula US, Hughes MS, Restifo NP, Raffeld M, Lee CC, Levy CL, Li YF, El-Gamil M, Schwarz SL, Laurencot C, Rosenberg SA. Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1. Journal of Clinical Oncology 2011;29(7) 917-924.
53 - Zhang G, Wang L, Cui H, Wang X, Zhang G, Ma J, Han H, He W, Wang W, Zhao Y, Liu C, Sun M, Gao B. Anti-melanoma activity of T cells redirected with a TCR-like chimeric antigen receptor. Sci Rep. 2014;4 3571.
54 - Hughes MS, Yu YY, Dudley ME, Zheng Z, Robbins PF, Li Y, Wunderlich J, Hawley RG, Moayeri M, Rosenberg SA, Morgan RA. Transfer of a TCR gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions. Human Gene Therapy 2005;16(4) 457-472.
55 - Willemsen R, Ronteltap C, Heuveling M, Debets R, Bolhuis R. Redirecting human CD4+T lymphocytes to the MHC class I-restricted melanoma antigen MAGE-A1 by TCR alphabeta gene transfer requires CD8alpha. Gene Therapy 2005; 12(2) 140-146.
56 - Morgan RA, Dudley ME, Wunderlich JR, Hughes MS, Yang JC, Sherry RM, Royal RE, Topalian SL, Kammula US, Restifo NP, Zheng Z, Nahvi A, de Vries CR, Rogers-Freezer LJ, Mavroukakis SA, Rosenberg SA. Cancer Regression in Patients After Transfer of Genetically Engineered Lymphocytes. Science. 2006;314(5796) 126-129.
57 - Morgan RA, Chinnasamy N, Abate-Daga D, Gros A, Robbins PF, Zheng Z, Dudley ME, Feldman SA, Yang JC, Sherry RM, Phan GQ, Hughes MS, Kammula US, Miller AD, Hessman CJ, Stewart AA, Restifo NP, Quezado MM, Alimchandani M, Rosenberg AZ, Nath A, Wang T, Bielekova B, Wuest SC, Akula N, McMahon FJ, Wilde S, Mosetter B, Schendel DJ, Laurencot CM, Rosenberg SA. Cancer regression and neurological toxicity following anti-MAGE-A3 TCR gene therapy. J Immunother 2013;36(2) 133-151.
58 - Coccoris M, Swart E, de Witte MA, van Heijst JW, Haanen JB, Schepers K, Schumacher TN. Long-term functionality of TCR-transduced T cells in vivo. Journal of Immunology 2008; 180(10) 6536-6543.
59 - Rosenberg SA, Yannelli JR, Yang JC, Topalian SL, Schwartzentruber DJ, Weber JS, Parkinson DR, Seipp CA, Einhorn JH, White DE. Treatment of patients with metastatic melanoma with autologous tumor-infiltrating lymphocytes and interleukin 2. Journal of the National Cancer Institute 1994;86(15) 1159–1166.
60 - Seliger B. Molecular mechanisms of MHC class I abnormalities and APM components in human tumors. Cancer Immunology Immunotherapy 2008;57(11) 1719-1726.
61 - Sigalotti L, Fratta E, Coral S, Tanzarella S, Danielli R, Colizzi F, Fonsatti E, Traversari C, Altomonte M, Maio M. Intratumor heterogeneity of cancer/testis antigens expression in human cutaneous melanoma is methylation-regulated and functionally reverted by 5-aza-2'-deoxycytidine. Cancer Research 2004;64(24) 9167-9171.
62 - Vitale M, Pelusi G, Taroni B, Gobbi G, Micheloni C, Rezzani R, Donato F, Wang X, Ferrone S. HLA class I antigen down-regulation in primary ovary carcinoma lesions: association with disease stage. Clinical Cancer Research 2005;11(1) 67-72.
63 - Coccoris M, Straetemans T, Govers C, Lamers C, Sleijfer S, Debets R. T cell receptor (TCR) gene therapy to treat melanoma: lessons from clinical and preclinical studies. Expert Opinion on Biological Therapy 2010;10(4) 547-562.
64 - Bendle GM, Linnemann C, Hooijkaas AI, Bies L, de Witte MA, Jorritsma A, Kaiser AD, Pouw N, Debets R, Kieback E, Uckert W, Song JY, Haanen JB, Schumacher TN. Lethal graft-versus-host disease in mouse models of T cell receptor gene therapy. Nature Medicine 2010; 16(5) 565-570.
65 - Cohen CJ, Zhao Y, Zheng Z, Rosenberg SA, Morgan RA. Enhanced antitumor activity of murine-human hybrid T-cell receptor (TCR) in human lymphocytes is associated with improved pairing and TCR/CD3 stability. Cancer Research 2006;66(17) 8878-8886.
66 - Kuball J, Dossett ML, Wolfl M, Ho WY, Voss RH, Fowler C, Greenberg PD. Facilitating matched pairing and expression of TCR chains introduced into human T cells. Blood 2007;109(6) 2331-2338.
67 - Eshhar Z, Waks T, Gross G, Schindler DG. Specific activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the gamma or zeta subunits of the immunoglobulin and T-cell receptors. Proceedings of the National Academy of Sciences of the United States of America 1993;90(2) 720-724.
68 - Cheadle EJ, Sheard V, Hombach, AA, Chmielewski M, Riet T, Berrevoets C, Schooten E, Lamers C, Abken H, Debets R, Gilham DE. Chimeric antigen receptors for T-cell based therapy. Methods Mol Biol 2012;907 645-666.
69 - Hombach A, Heuser C, Sircar R, Tillmann T, Diehl V, Kruis W, Pohl C, Abken H. T cell targeting of TAG72+tumor cells by a chimeric receptor with antibody-like specificity for a carbohydrate epitope. Gastroenterol 1997;113(4), 1163 – 1170.
70 - Hombach A, Abken H. Costimulation tunes tumor-specific activation of redirected T cells in adoptive immunotherapy,” Cancer Immunology Immunotherapy 2007;56(5)731-737.
71 - A service of the U.S. National Institutes of Health. Trial ID: NCT00586391
72 - A service of the U.S. National Institutes of Health. Trial ID: NCT00709033
73 - Hombach AA, Abken H. Costimulation by chimeric antigen receptors revisited the T cell antitumor response benefits from combined CD28-OX40 signalling. International Journal of Cancer 2011;129(12) 2935-2944.
74 - Hombach AA, Chmielewski M, Rappl G, Abken H. Adoptive immunotherapy with redirected T cells produces CCR7-cells that are trapped in the periphery and benefit from combined CD28-OX40 costimulation. Hum Gene Ther 2013;24(3) 259-269.
75 - Bridgeman JS, Hawkins RE, Hombach AA, Abken H, Gilham DE.Building better chimeric antigen receptors for adoptive T cell therapy. Current Gene Therapy 2010; 10(2) 77-90.
76 - Gilham DE, Debets R, Pule M, Hawkins RE, Abken H. CAR-T cells and solid tumors: tuning T cells to challenge an inveterate foe. Trends in Molecular Medicine 2012;18(7) 377-384.
77 - Reinhold U, Liu L, Lüdtke-Handjery HC, Heuser C, Hombach A, Wang X, Tilgen W, Ferrone S, Abken H. Specific lysis of melanoma cells by receptor grafted T cells is enhanced by anti-idiotypic monoclonal antibodies directed to the scFv domain of the receptor. The Journal of Investigative Dermatology 1999;112(5) 744-750.
78 - Burns WR, Zhao Y, Frankel TL, Hinrichs CS, Zheng Z, Xu H, Feldman SA, Ferrone S, Rosenberg SA, Morgan RA. A high molecular weight melanoma-associated antigen-specific chimeric antigen receptor redirects lymphocytes to target human melanomas. Cancer Research 2010;70(8) 3027-3033.
79 - Schmidt P, Kopecky C, Hombach A, Zigrino P, Mauch C, Abken H. Eradication of melanomas by targeted elimination of a minor subset of tumor cells. Proceedings of the National Academy of Sciences of the United States of America 2011;108(6) 2474-2479.
80 - Yvon E, Del Vecchio M, Savoldo B, Hoyos V, Dutour A, Anichini A, Dotti G, Brenner MK. Immunotherapy of metastatic melanoma using genetically engineered GD2-specific T cells. Clinical Cancer Research 2009;15(18) 5852-5860.
81 - Lo AS, Ma Q, Liu DL, Junghans RP. Anti-GD3 chimeric sFv-CD28/T-cell receptor zeta designer T cells for treatment of metastatic melanoma and other neuroectodermal tumors. Clinical Cancer Research 2010;16(10) 2769-2780.
82 - Kalos M, Levine BL, Porter DL, Katz S, Grupp SA, Bagg A, June CH. T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia. Science Translational Medicine 2011;3(95) 95ra73.
83 - Porter DL, Levine BL, Kalos M, Bagg A, June CH. Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. The New England Journal of Medicine 2011;365(8) 725-733.
84 - Lamers CH, Sleijfer S, Vulto AG, Kruit WH, Kliffen M, Debets R, Gratama JW, Stoter G, Oosterwijk E. Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX: first clinical experience. Journal of Clinical Oncology 2006; 24(3) 20-22.
85 - Brentjens R, Yeh R, Bernal Y, Riviere I, Sadelain M. Treatment of chronic lymphocytic leukemia with genetically targeted autologous T cells: case report of an unforeseen adverse event in a phase I clinical trial. Molecular Therapy 2010;18(4) 666-668.
86 - Morgan RA, Yang JC, Kitano M, Dudley ME, Laurencot CM, Rosenberg SA. Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Molecular Therapy 2010;8(4) 843-851.
87 - Hawkins RE, Gilham DE, Debets R, Eshhar Z, Taylor N, Abken H, Schumacher TN, ATTACK Consortium. Development of adoptive cell therapy for cancer: a clinical perspective. Human Gene Therapy 2010;21(6) 665-672.
88 - Büning H, Uckert W, Cichutek K, Hawkins RE, Abken H. Do CARs need a driver's license? Adoptive cell therapy with chimeric antigen receptor-redirected T cells has caused serious adverse events. Human Gene Therapy 2010;21(9) 1039-1042.
89 - Zhou BB, Zhang H, Damelin M, Geles KG, Grindley JC, Dirks PB. Tumour-initiating cells: challenges and opportunities for anticancer drug discovery. Nature Reviews Drug Discovery 2009;8(10) 806-823.
90 - Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proceedings of the National Academy of Sciences of the United States of America 2003;100(7) 3983-3988.
91 - Dalerba P, Dylla SJ, Park IK, Liu R, Wang X, Cho RW, Hoey T, Gurney A, Huang EH, Simeone DM, Shelton AA, Parmiani G, Castelli C, Clarke MF. Phenotypic characterization of human colorectal cancer stem cells. Proceedings of the National Academy of Sciences of the United States of America 2007;104(24) 10158-10163.
92 - Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C, Squire J, Dirks PB. Identification of a cancer stem cell in human brain tumors. Cancer Research 2003;63(18) 5821-5828.
93 - Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M, Peschle C, De Maria R. Identification and expansion of human colon-cancer-initiating cells. Nature 2007;445(7123) 111-115.
94 - Li C, Heidt DG, Dalerba P, Burant CF, Zhang L, Adsay V, Wicha M, Clarke MF, Simeone DM. Identification of pancreatic cancer stem cells. Cancer Research 2007;67(3) 1030-1037.
95 - Schatton T, Murphy GF, Frank NY, Yamaura K, Waaga-Gasser AM, Gasser M, Zhan Q, Jordan S, Duncan LM, Weishaupt C, Fuhlbrigge RC, Kupper TS, Sayegh MH, Frank MH. Identification of cells initiating human melanomas. Nature. 2008;451(7176) 345-349.
96 - Zabierowski SE, Herlyn M. Melanoma stem cells: the dark seed of melanoma. Journal of Clinical Oncology 2008;26(17) 2890-2894.
97 - Fang D, Nguyen TK, Leishear K, Finko R, Kulp AN, Hotz S, Van Belle PA, Xu X, Elder DE, Herlyn M. A tumorigenic subpopulation with stem cell properties in melanomas. Cancer Research 2005;65(20) 9328-9337.
98 - Boiko AD, Razorenova OV, van de Rijn M, Swetter SM, Johnson DL, Ly DP, Butler PD, Yang GP, Joshua B, Kaplan MJ, Longaker MT, Weissman IL. Human melanoma-initiating cells express neural crest nerve growth factor receptor CD271. Nature 2010;466(7302) 133-137.
99 - Quintana E, Shackleton M, Sabel MS, Fullen DR, Johnson TM, Morrison SJ. Efficient tumour formation by single human melanoma cells. Nature 2008;456(7222) 593-598.
100 - Quintana E, Shackleton M, Foster HR, Fullen DR, Sabel MS, Johnson TM, Morrison SJ. Phenotypic Heterogeneity among Tumorigenic Melanoma Cells from Patients that Is Reversible and Not Hierarchically Organized. Cancer Cell 2010;18(5) 510-523.
101 - Shakhova O, Zingg D, Schaefer SM, Hari L, Civenni G, Blunschi J, Claudinot S, Okoniewski M, Beermann F, Mihic-Probst D, Moch H, Wegner M, Dummer R, Barrandon Y, Cinelli P, Sommer L. Sox10 promotes the formation and maintenance of giant congenital naevi and melanoma. Nat Cell Biol 2012;14(8) 882-890.
102 - Schlaak M, Schmidt P, Bangard C, Kurschat P, Mauch C, Abken H. Regression of metastatic melanoma in a patient by antibody targeting of cancer stem cells. Oncotarget 2012;3(1) 22-30.
103 - Pinc A, Somasundaram R, Wagner C, Hörmann M, Karanikas G, Jalili A, Bauer W, Brunner P, Grabmeier-Pfistershammer K, Gschaider M, Lai CY, Hsu MY, Herlyn M, Stingl G, Wagner SN. Targeting CD20 in melanoma patients at high risk of disease recurrence. Mol Ther 2012;20(5) 1056-1062.
104 - Chmielewski M, Rappl G, Hombach AA, Abken H. T cells redirected by a CD3ζ chimeric antigen receptor can establish self-antigen-specific tumour protection in the long term. Gene Ther. 2013;20(2) 177-186.
105 - Kershaw MH, Westwood JA, Parker LL, Wang G, Eshhar Z, Mavroukakis SA, White DE, Wunderlich JR, Canevari S, Rogers-Freezer L, Chen CC, Yang JC, Rosenberg SA, Hwu P.A phase I study on adoptive immunotherapy using gene-modified T cells for ovarian cancer. Clin Cancer Res 2006;12(20.1) 6106-6115.
106 - Brentjens RJ, Davila ML, Riviere I, Park J, Wang X, Cowell LG, Bartido S, Stefanski J, Taylor C, Olszewska M, Borquez-Ojeda O, Qu J, Wasielewska T, He Q, Bernal Y, Rijo IV, Hedvat C, Kobos R, Curran K, Steinherz P, Jurcic J, Rosenblat T, Maslak P, Frattini M, Sadelain M. CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci Transl Med. 2013;5(177) 177ra38.
107 - Kaneko S, Mastaglio S, Bondanza A, Ponzoni M, Sanvito F, Aldrighetti L, Radrizzani M, La Seta-Catamancio S, Provasi E, Mondino A, Nagasawa T, Fleischhauer K, Russo V, Traversari C, Ciceri F, Bordignon C, Bonini C. IL-7 and IL-15 allow the generation of suicide gene-modified alloreactive self-renewing central memory human T lymphocytes. Blood 2009; 113(5) 1006-1015.
108 - Suhoski MM, Golovina TN, Aqui NA, Tai VC, Varela-Rohena A, Milone MC, Carroll RG, Riley JL, June CH. Engineering artificial antigen-presenting cells to express a diverse array of co-stimulatory molecules. Mol Ther 2007;15(5) 981-988.
109 - Vera JF, Brenner LJ, Gerdemann U, Ngo MC, Sili U, Liu H, Wilson J, Dotti G, Heslop HE, Leen AM, Rooney CM. Accelerated production of antigen-specific T cells for preclinical and clinical applications using gas-permeable rapid expansion cultureware (G-Rex). J Immunother 2010;33(3) 305-315.
110 - Meidenbauer N, Marienhagen J, Laumer M, Vogl S, Heymann J, Andreesen R, Mackensen A. Survival and tumor localization of adoptively transferred Melan-A-specific T cells in melanoma patients. Journal of Immunology 2003;170(4) 2161-2169.
111 - Griffith KD, Read EJ, Carrasquillo JA, Carter CS, Yang JC, Fisher B, Aebersold P, Packard BS, Yu MY, Rosenberg SA. In vivo distribution of adoptively transferred indium-111-labeled tumor infiltrating lymphocytes and peripheral blood lymphocytes in patients with metastatic melanoma. Journal of the National Cancer Institute 1989;81(22) 1709-1717.
112 - Fisher B, Packard BS, Read EJ, Carrasquillo JA, Carter CS, Topalian SL, Yang JC, Yolles P, Larson SM, Rosenberg SA. Tumor localization of adoptively transferred indium-111 labeled tumor infiltrating lymphocytes in patients with metastatic melanoma. Journal of Clinical Oncology 1989;7(2) 250-261.
113 - Peng W, Ye Y, Rabinovich BA, Liu C, Lou Y, Zhang M, Whittington M, Yang Y, Overwijk WW, Lizée G, Hwu P. Transduction of tumor-specific T cells with CXCR2 chemokine receptor improves migration to tumor and antitumor immune responses. Clinical Cancer Research 2010;16(22) 5458-5468.
114 - Li Y, Liu S, Hernandez J, Vence L, Hwu P, Radvanyi L.MART-1-specific melanoma tumor-infiltrating lymphocytes maintaining CD28 expression have improved survival and expansion capability following antigenic restimulation in vitro. Journal of Immunology 2010;184(1) 452-465.
115 - Hernandez-Chacon JA, Li Y, Wu RC, Bernatchez C, Wang Y, Weber JS, Hwu P, Radvanyi LG. Costimulation through the CD137/4-1BB pathway protects human melanoma tumor-infiltrating lymphocytes from activation-induced cell death and enhances antitumor effector function. Journal of Immunology 2011;34(3) 236-250.
116 - Offringa R. Antigen choice in adoptive T-cell therapy of cancer. Current Opinion in Immunology 2009;21(2) 190-199.
117 - Overwijk WW, Theoret MR, Finkelstein SE, Surman DR, de Jong LA, Vyth-Dreese FA, Dellemijn TA, Antony PA, Spiess PJ, Palmer DC, Heimann DM, Klebanoff CA, Yu Z, Hwang LN, Feigenbaum L, Kruisbeek AM, Rosenberg SA, Restifo NP. Tumor regression and autoimmunity after reversal of a functionally tolerant state of self-reactive CD8+T cells. The Journal of Experimental Medicine 2003;198(4) 569-580.
118 - Spiotto MT, Rowley DA, and Schreiber H. “Bystander” elimination of antigen loss variants in established tumors. Nature Medicine 2004; 10(3) 294-298.
119 - Schüler T, Blankenstein T. Cutting edge: CD8+effector T cells reject tumors by direct antigen recognition but indirect action on host cells. Journal of Immunology 2003;170(9) 4427-4431.
120 - Stewart-Jones G, Wadle A, Hombach A, Shenderov E, Held G, Fischer E, Kleber S, Nuber N, Stenner-Liewen F, Bauer S, McMichael A, Knuth A, Abken H, Hombach AA, Cerundolo V, Jones EY, Renner C. Rational development of high-affinity T-cell receptor-like antibodies. Proceedings of the National Academy of Sciences of the United States of America 2009;106(14) 5784-5788.
121 - Willemsen RA, Debets R, Hart E, Hoogenboom HR, Bolhuis RL, Chames P. A phage display selected fab fragment with MHC class I-restricted specificity for MAGE-A1 allows for retargeting of primary human T lymphocytes. Gene Therapy 2001;8(21) 1601-1608.
122 - Chmielewski M, Hombach A, Heuser C, Adams GP, Abken H.T cell activation by antibody-like immunoreceptors: increase in affinity of the single-chain fragment domain above threshold does not increase T cell activation against antigen-positive target cells but decreases selectivity. Journal of Immunology 2004; 173(12) 7647-7653.
123 - Chmielewski M, Hombach AA, Abken H. CD28 cosignalling does not affect the activation threshold in a chimeric antigen receptor-redirected T-cell attack. Gene Therapy 2011;18(1) 62-72.
124 - Pule MA, Savoldo B, Myers GD, Rossig C, Russell HV, Dotti G, Huls MH, Liu E, Gee AP, Mei Z, Yvon E, Weiss HL, Liu H, Rooney CM, Heslop HE, Brenner MK. Virus-specific T cells engineered to coexpress tumor-specific receptors: persistence and antitumor activity in individuals with neuroblastoma Nature Medicine 2008;14(11) 1264-1270.
125 - Antony PA, Piccirillo CA, Akpinarli A, Finkelstein SE, Speiss PJ, Surman DR, Palmer DC, Chan CC, Klebanoff CA, Overwijk WW, Rosenberg SA, Restifo NP. CD8+T cell immunity against a tumor/self-antigen is augmented by CD4+T helper cells and hindered by naturally occurring T regulatory cells. Journal of Immunology 2005;174(5) 2591-2601.
126 - Kofler DM, Chmielewski M, Rappl G, Hombach A, Riet T, Schmidt A, Hombach AA, Wendtner CM, Abken H. CD28 costimulation Impairs the efficacy of a redirected t-cell antitumor attack in the presence of regulatory t cells which can be overcome by preventing Lck activation. Mol Ther 2011;19(4) 760-767.
127 - [127]Leach DR, Krummel MF, Allison JP. Enhancement of antitumor immunity by CTLA-4 blockade. Science 1996;271(5256) 1734-1736.
128 - [128]Pedicord VA, Montalvo W, Leiner IM, Allison JP. Single dose of anti-CTLA-4 enhances CD8+T-cell memory formation, function, and maintenance. Proc Natl Acad Sci USA 2011;108(1) 266-271.
129 - Abate-Daga D, Hanada K, Davis JL, Yang JC, Rosenberg SA, Morgan RA. Expression profiling of TCR-engineered T cells demonstrates overexpression of multiple inhibitory receptors in persisting lymphocytes. Blood 2013;122(8) 1399-1410.
130 - Moon EK, Wang LC, Dolfi DV, Wilson CB, Ranganathan R, Sun J, Kapoor V, Scholler J, Puré E, Milone MC, June CH, Riley JL, Wherry EJ, Albelda SM. Multifactorial T-cell Hypofunction That Is Reversible Can Limit the Efficacy of Chimeric Antigen Receptor-Transduced Human T cells in Solid Tumors. Clin Cancer Res 2014;20(16) 4262-4273.
131 - Mackensen A, Meidenbauer N, Vogl S, Laumer M, Berger J, Andreesen R. Phase I study of adoptive T-cell therapy using antigen-specific CD8+T cells for the treatment of patients with metastatic melanoma. Journal of Clinical Oncology 2006;24(31) 5060-5069.
132 - Lozupone F, Rivoltini L, Luciani F, Venditti M, Lugini L, Cova A, Squarcina P, Parmiani G, Belardelli F, Fais S. Adoptive transfer of an anti-MART-1(27-35)-specific CD8+T cell clone leads to immunoselection of human melanoma antigen-loss variants in SCID mice. European Journal of Immunology 2003;33(2) 556-566.
133 - Becker JC, Varki N, Gillies SD, Furukawa K, Reisfeld RA. An antibody-interleukin 2 fusion protein overcomes tumor heterogeneity by induction of a cellular immune response,” Proceedings of the National Academy of Sciences of the United States of America 1996;93(15) 7826-7831.
134 - Chmielewski M, Kopecky C, Hombach AA, Abken H. IL-12 release by engineered T cells expressing chimeric antigen receptors can effectively Muster an antigen-independent macrophage response on tumor cells that have shut down tumor antigen expression. Cancer Research 2011;71(17) 5697-5706.
135 - Boni A, Cogdill AP, Dang P, Udayakumar D, Njauw CN, Sloss CM, Ferrone CR, Flaherty KT, Lawrence DP, Fisher DE, Tsao H, Wargo JA. Selective BRAFV600E inhibition enhances T-cell recognition of melanoma without affecting lymphocyte function. Cancer Research 2010;70(13) 5213-5219.
136 - Wilmott JS, Long GV, Howle JR, Haydu LE, Sharma RN, Thompson JF, Kefford RF, Hersey P, Scolyer RA. Selective BRAF inhibitors induce marked T-cell infiltration into human metastatic melanoma. Clinical Cancer Research 2012;18(5) 1386-1394.
137 - Pegram HJ, Jackson JT, Smyth MJ, Kershaw MH, Darcy PK. Adoptive transfer of gene-modified primary NK cells can specifically inhibit tumor progression in vivo. Journal of Immunology 2008;181(5) 3449-3455.
138 - Kruschinski A, Moosmann A, Poschke I, Norell H, Chmielewski M, Seliger B, Kiessling R, Blankenstein T, Abken H, Charo J. Engineering antigen-specific primary human NK cells against HER-2 positive carcinomas,” Proceedings of the National Academy of Sciences of the United States of America 2008;105(45) 17481-17486.
139 - Boissel L, Betancur-Boissel M, Lu W, Krause DS, Van Etten RA, Wels WS, Klingemann H. Retargeting NK-92 cells by means of CD19-and CD20-specific chimeric antigen receptors compares favorably with antibody-dependent cellular cytotoxicity. Oncoimmunology 2013;2(10) e26527.
140 - Jiang H, Zhang W, Shang P, Zhang H, Fu W, Ye F, Zeng T, Huang H, Zhang X, Sun W, Man-Yuen Sze D, Yi Q, Hou J. Transfection of chimeric anti-CD138 gene enhances natural killer cell activation and killing of multiple myeloma cells. Mol Oncol 2014;8(2) 297-310.
141 - Boissel L, Betancur M, Wels WS, Tuncer H, Klingemann H. Transfection with mRNA for CD19 specific chimeric antigen receptor restores NK cell mediated killing of CLL cells. Leuk Res 2009; 33(9), 1255-1259.
142 - Klingemann HG. Cellular therapy of cancer with natural killer cells-where do we stand? Cytotherapy 2013;15(10) 1185-1194.
143 - Müller T, Uherek C, Maki G, Chow KU, Schimpf A, Klingemann HG, Tonn T, Wels WS. Expression of a CD20-specific chimeric antigen receptor enhances cytotoxic activity of NK cells and overcomes NK-resistance of lymphoma and leukemia cells. Cancer Immunol Immunother 2008;57(3) 411-423.
144 - Chu J, Deng Y, Benson DM, He S, Hughes T, Zhang J, Peng Y, Mao H, Yi L, Ghoshal K, He X, Devine SM, Zhang X, Caligiuri MA, Hofmeister CC, Yu J. CS1-specific chimeric antigen receptor (CAR)-engineered natural killer cells enhance In Vitro and In Vivo anti-tumor activity against human multiple myeloma. Leukemia 2014;28(4) 917-927.
145 - Uherek C, Tonn T, Uherek B, Becker S, Schnierle B, Klingemann HG, Wels W. Retargeting of natural killer-cell cytolytic activity to ErbB2-expressing cancer cells results in efficient and selective tumor cell destruction. Blood 2002;100(4) 1265-1273.
146 - Tavri S, Jha P, Meier R, Henning TD, Müller T, Hostetter D, Knopp C, Johansson M, Reinhart V, Boddington S, Sista A, Wels WS, Daldrup-Link HE. Optical imaging of cellular immunotherapy against prostate cancer. Mol Imaging2009;8(1) 15-26.
147 - Esser R, Müller T, Stefes D, Kloess S, Seidel D, Gillies SD, Aperlo-Iffland C, Huston JS, Uherek C, Schönfeld K, Tonn T, Huebener N, Lode HN, Koehl U, Wels WS. NK cells engineered to express a GD2-specific antigen receptor display built-in ADCC-like activity against tumour cells of neuroectodermal origin. J Cell Mol Med 2012;16(3) 569-581.
148 - Imai C, Iwamoto S, Campana D. Genetic modification of primary natural killer cells overcomes inhibitory signals and induces specific killing of leukemic cells. Blood 2005;106(1) 376-383.
149 - Altvater B, Landmeier S, Pscherer S, Temme J, Schweer K, Kailayangiri S, Campana D, Juergens H, Pule M, Rossig C. 2B4 (CD244) signaling by recombinant antigen-specific chimeric receptors costimulates natural killer cell activation to leukemia and neuroblastoma cells. Clin Cancer Res2009;15(15) 4857-4866.
150 - Sahm C, Schönfeld K, Wels WS. Expression of IL-15 in NK cells results in rapid enrichment and selective cytotoxicity of gene-modified effectors that carry a tumor-specific antigen receptor. Cancer Immunol Immunother 2012;61(9) 1451-1461.