The T-cell growth factor interleukin (IL)-2 can activate endogenous tumor-reactive cells, and reproducibly mediate the regression of advanced metastatic melanoma and kidney cancer. In a consecutive series of 409 patients treated at the Surgery Branch of the National Cancer Institute (NCI), between 1985 and 1996, high-dose bolus intravenous IL-2 administration produced complete and durable regressions of metastatic melanoma and renal cell cancer in 6.6% and 9.3% of patients, respectively [9,10]. Responses were seen at all sites of disease, and more than 80% of complete responses appeared durable and were ongoing after a median follow-up of 7 years at the time of publication. IL-2 alone does not appear sufficient to induce regression of other solid cancers. The US Food and Drug Administration (FDA) approved the use of IL-2 for the treatment of metastatic renal cancer in 1992 and metastatic melanoma in 1998, based on the ability of IL-2 to mediate durable complete responses. For the same reason, IL-2 therapy should be the first-line treatment for patients with metastatic renal cancer and metastatic melanoma.

Experience with the administration of IL-2 has resulted in treatment-related mortalities of less than 1%. Toxicities occur owing to a capillary leak syndrome, and can be safely treated with appropriate monitoring and judicious fluid resuscitation [11]. Organ-specific autoimmunity seen in melanoma patients treated with IL-2, such as delayed-onset vitiligo in approximately 20% of patients and the development of autoimmune thyroiditis in approximately 55%, has been associated with the likelihood of cancer regression [12]. Of interest, patients with renal cell carcinoma do not develop vitiligo with IL-2, and autoimmune thyroid is seen less commonly in these patients, arguing for activation of a different subset of T cells in melanoma patients, able to recognize antigens expressed on tumor and on normal melanocytes (mainly MART-1, gp100, and Tyrosinase) [10].

Interferon-α2b (IFN-α2b), an important mediator of antiviral immunity, has also been used for the treatment of patients with melanoma and renal cell cancer. The role of IFN-α2b for the adjuvant treatment of patients at high risk of recurrence after definitive surgery is controversial, since improvements in recurrence-free survival have translated into little impact on overall survival. Long-term administration of a pegylated formulation, expected to maintain maximum exposure to IFN-α2b with less frequent subcutaneous injections than with the unpegylated formulation, has recently led to similar results in a large randomized control trial of patients with node-positive melanoma (stage III) [13,14]. In this trial, the relapse-free survival was improved by 9.3 months (34.8 months vs 25.5 months), without benefit in overall survival. The FDA approved pegylated IFN-α2b (Sylatron) on March 29, 2011 for the treatment of melanoma patients with microscopic or gross nodal involvement.

Activated T cells express surface inhibitory molecules that, when bound by their ligands, are capable of inhibiting T-cell activity. Monoclonal antibodies directed against these surface inhibitory molecules have been studied as immunotherapeutic regents. The cytotoxic T-lymphocyte–associated 4 molecule (CTLA-4, CD152), part of the immunoglobulin-like family of surface proteins, is one of the inhibitory molecules expressed at the surface of activated T cells. Two fully human IgG monoclonal antibodies recognizing CTLA-4, ipilimumab (MDX-010) and tremelimumab (CP-675,206), have been tested, alone or in combination, in phase 2/3 trials. These antibodies are designed to prevent the binding of CTLA-4 to its ligand B7, mainly expressed on immature antigen-presenting cells and tumor cells [15].

The first demonstration of the ability of ipilimumab to mediate tumor regression in 2003 reported objective regressions in 3 of 13 patients with metastatic melanoma [16]. An updated summary of consecutive cohorts of 179 patients with metastatic melanoma revealed an overall response rate ranging from 13% to 25%, including 6% to 17% durable complete responses [17]. The highest response rates were seen in patients given ipilimumab in combination with high-dose IL-2. In this cohort, 6 of 36 (17%) patients enjoy ongoing complete responses after 7 years of median follow-up. It is interesting that prior response to IL-2 was not correlated with the likelihood of response to ipilimumab, pointing to a different quality of interaction between melanoma and the host immune system using these two agents alone. Important but delayed responses to treatment were observed in some patients.

Immune-related adverse events were more frequently observed in responders than in nonresponders, and could be severe. Approximately 35% of patients developed Grade 3 and 4 immune-related toxicities, the most common being enterocolitis in 17%, followed by hypophysitis and dermatitis in 9% and 6%, respectively. Other less common side effects included hepatitis, nephritis, uveitis, and arthritis. With the exception of hypophysitis with hypopituitarism, immune-related complications were usually reversible with systemic and topical corticosteroid treatment. The addition of anti–tumor necrosis factor α (infliximab) monoclonal antibodies to systemic corticosteroids has been successfully used to treat patients with severe colitis. As reported in a recent literature review, a colectomy may be life-saving for some patients—as much as 12% in one series—who develop bleeding or perforation from colitis unresponsive to medical therapy [18].

The effectiveness of ipilimumab as second-line treatment for patients with advanced melanoma has now been confirmed in a large double-blinded, randomized, multi-institutional phase 3 trial [19]. A total of 676 HLA-A*0201 patients were randomized 3:1:1 to receive ipilimumab plus a gp100 vaccine, ipilimumab alone, or the gp100 vaccine alone. The overall median survival was equivalent in both groups of patients who received ipilimumab, approximately 10 months versus 6.4 months for the group who received the gp100 vaccine alone. Objective responses were seen in 38 patients among the 540 who received ipilimumab (7%), with 3 complete responses (0.5%). Immune-related side effects were seen at comparable rates to what has been described above, and 4 patients died following bowel perforation, due to ipilimumab-induced colitis. The addition of the vaccine did not confer a survival benefit or lead to unexpected side effects. Based on the results of this trial, the FDA approved ipilimumab (Yervoy) for the treatment of unresectable or metastatic melanoma on March 25, 2011. Ongoing trials are now assessing the efficacy anti–CTLA-4 in combination with other biological and cytotoxic agents, notably dacarbazine [20].

The efficacy of ipilimumab has been tested for other solid malignancies. In a nonrandomized phase 2 study of 40 patients with metastatic renal cell cancer treated with ipilimumab alone, 5 patients had a partial response [21]. A single or 2 doses of ipilimumab has been associated with a decrease of 50% or more of the prostate-specific antigen (PSA) level in 2 of 12 patients with metastatic hormone-refractory prostate cancer patients [22]. Of 27 patients with unresectable or metastatic pancreatic adenocarcinoma treated with ipilimumab, only one experienced a mixed response [23].

Tremelimumab, the second fully humanized anti–CTLA-4 monoclonal antibody, has been less studied than ipilimumab. In the most recent and largest phase 2 trial in patients with advanced melanoma treated with tremelimumab, a response rate of 6.6% in 246 patients was reported, without complete responses, 2 treatment-related deaths, and a median survival of 10 months [24]. In heavily pretreated metastatic colorectal cancer patients, 1 of 45 partial but sustained response was reported [25].

Programmed death 1 (PD-1, CD279) is another inhibitory molecule expressed at the surface of activated T cells after repeated encounter with antigen. It belongs to the same family of surface molecules as CTLA-4, but binds different ligands and provides distinct intracellular signaling that leads to shutdown of T-cell effector function. Tumor cells and antigen-presenting cells found in tumors can express a high level of PD-L1, the main PD-1 ligand (also called B7-H1), and this has been associated with poor prognosis in renal and ovarian cancer [26,27]. Interrupting the interaction between PD-1 and its ligand using the fully human IgG4 monoclonal antibody MDX-1106 has been reported to mediate cancer regression in a phase 1, dose-escalation trial [28]. Objective response was documented in 3 of 39 patients, one each with melanoma, renal cancer, and colorectal cancer.

Overall, nonspecific modulation of immunity, either to promote activation or to block inhibition of effector T cells, can mediate tumor regression mainly in a subset of patients with metastatic melanoma and renal cancer. Although occasional tumor responses have been observed for other solid cancers, melanoma and renal cancer appear exceptional in their ability to harbor endogenous antitumor cells of sufficient avidity and in sufficient numbers to respond to nonspecific immunomodulators. It remains to be seen if combination of standard chemotherapy with immunomodulators such as anti–CTLA-4 and anti-PD1, strategies currently being tested, will expand the use of these agents to other solid tumors. Investigations are under way to evaluate other general immune modulators for solid cancer treatment. IL-15 is under investigation at the NCI for patients with melanoma. The rationale behind using IL-15 instead of IL-2 is to promote the expansion of a pool of effector-memory T cells and to avoid preferential expansion of regulatory T cells, because the later constitutively express the high-affinity receptor chain for IL-2. IL-21, another T-cell–stimulating cytokine, was reported to mediate objective response in 22% of patients with metastatic melanoma [29]. IL-12, a cytokine that activates APCs, T cells, and the natural killer subset of lymphocytes, had shown good tumor effect in animal models; however, attempts to give a therapeutic dose of IL-12 systemically in humans have been limited by toxicities [30]. Current efforts are focused on strategies to deliver IL-12 at the tumor site in order to avoid systemic toxicities [31].

Therapeutic cancer vaccines have consistently shown low efficacy in the treatment of metastatic cancer. Anecdotal responses, in melanoma as well as in other solid tumors, have been reported. Although the success of any vaccination strategy remains dependent on the choice of the appropriate tumor antigen to target, improvement may be achieved by investigating many aspects of the vaccination strategy and effect. These approaches include assessing the frequency of T-cell precursors specific for chosen tumor antigens, defining the quality and amplitude of the cellular immune response to be mounted by a given vaccine strategy, verifying if newly primed T cells can reach and infiltrate the tumor rather than preferentially accumulate at the vaccination site and in the immediate draining lymph nodes, and testing strategies to overcome the immunosuppressive tumor microenvironment.

In 1986 it was demonstrated that murine sarcoma and colon adenocarcinoma transplanted in non-immunized syngeneic mice harbored TIL that could be expanded in vitro with IL-2 and would mediate regression of disseminated tumors after adoptive transfer (Rosenberg SA and colleagues, Science 1986 [51]). The adoptive transfer of TIL obtained from human melanomas was first shown to mediate regression of autologous metastatic melanoma in 1988 [52], but decisive improvement in efficacy came in 2002 with the introduction of an immunodepleting preparative regimen given before TIL infusion [53]. This approach could result in clonal repopulation of patients’ circulating lymphocytes with antitumor activity [54]. This lymphodepleting preparative regimen was shown to contribute to the antitumor activity of the transferred TIL primarily by depleting endogenous regulatory cells and by depleting endogenous lymphocytes that competed with the transferred cells for growth-promoting homeostatic cytokines such as IL-7 and IL-15. A schematic description of TIL therapy is illustrated in Fig. 1.

Fig. 1 The generation of tumor-infiltrating lymphocytes for adoptive cell transfer therapy. A tumor is excised from a patient with metastatic melanoma, and cultures of tumor-infiltrating lymphocytes are grown from fragments or cell suspensions. Lymphocytes with tumor reactivity are expanded to large numbers (>10¹⁰ cells) for reinfusion into the patient preconditioned by a lymphodepleting preparative regimen. IL-2, interleukin-2; TIL, tumor-infiltrating lymphocytes.

Three sequential ACT trials performed in the Surgery Branch, NCI, on 93 patients using autologous TIL harvested from metastatic melanoma patients infused with IL-2 after preconditioning immune suppression, are summarized in Table 1. The median potential follow-up of these trials was 69 months. Increasing the level of immune suppression using total body irradiation combined with lymphodepleting chemotherapy before TIL infusion was associated with a higher overall and complete response rate. The objective response rate by RECIST criteria reached 72% with maximum immune suppression, including 40% of patients with complete tumor eradication. Of the 20 complete responders enrolled in these 3 trials only one has relapsed, with all others in ongoing complete response beyond 3 to 7 years. Nonhematological grade 3 and 4 toxicities observed in the cohort of patients receiving the nonmyeloablative regimen were febrile neutropenia in 37% and intubation for dyspnea in 9%. Adding total body irradiation at high dose led to more intubation for somnolence, but comparable rate of adverse events otherwise. In 93 patients treated, one mortality was observed consequent to an unrecognized diverticular abscess.

Not all patients with metastatic melanoma are candidates for surgical excision of a tumor metastasis necessary to generate TIL. In addition, TIL with demonstrable antitumor activity have rarely been generated from human tumors other than melanoma. For these reasons, techniques have been developed to genetically modify peripheral lymphocytes to express a receptor able to recognize tumor antigens. Gammaretroviral vectors have provided an efficient and safe way to introduce new genes into lymphocytes.

Two types of receptors can be introduced into T cells to redirect effector T-cell specificity to tumor antigens. The first is a conventional TCR comprising two chains (α and β) that recognize peptides presented by MHC molecules. Thus, these TCRs can recognize antigens only on specific human leukocyte antigen haplotypes (HLA). The first vectors were designed to recognize peptides presented by HLA-A*0201, one of the most commonly expressed HLA in humans. The second type of receptor that recognizes tumor antigens is called chimeric antigen receptor (CAR). A CAR is a fusion protein that links the variable portions of the heavy and light chains of an antibody to the intracellular signaling domains of a TCR. Introduced into a T cell, CAR enable the lymphocyte to recognize tridimensional proteins found at the surface of tumor cells without MHC restriction, rather than a short peptide nested in MHC molecules recognized by conventional TCR. By combining the antigen specificity of an antibody and the cytotoxic properties of a T cell in an HLA-unrestricted manner, CAR can be resistant to tumor-immune evasion mechanisms, such as downregulation of MHC molecules and failure to process antigens to the cell surface. Selected TCR and CAR expression vectors optimized at the Surgery Branch are summarized in Fig. 2. In the United States, all clinical trials using gene transfer technology are reviewed by the National Institutes of Health Office of Biotechnology (OBA) Activities. A list of gene therapy protocols can be found on the OBA Web site (http://oba.od.nih.gov), and on the Clinical Trials Web site (www.clinicaltrials.gov).

Fig. 2 Gammaretroviral vectors and receptors used to engineer anticancer lymphocytes. T cells can be engineered with two classes of receptors that are capable of recognizing tumor-associated antigens. (A) T-cell receptors (TCR) require coordinated expression of an α and β chain, which is facilitated by the use of a 2A fusion protein. (B) Chimeric antigen receptors (CAR) are artificially constructed hybrid proteins combining the antigen specificity of the variable region of the heavy (V_H) and light (V_L) chain of an antibody (scFv) linked to T-cell signaling domains, such as CD3ζ and CD28. Vector-specific cis-acting sequences are the long terminal repeat (LTR) that contains the enhancer, promoter, and polyadenylation sites, splice donor (SD) and splice acceptor (SA) sequences, and packaging signal (ψ).

Adoptive transfer of TIL is the most effective therapy reported thus far for patients with metastatic melanoma. A simplified method of generating TIL, allowing for the treatment of additional patients in a shorter time frame, is currently being clinically evaluated [74]. Other institutions have now begun to treat patients with metastatic melanoma with adoptive transfer of TIL. At the M.D. Anderson Cancer Center, approximately a 50% objective rate was seen in 30 patients receiving TIL selected for tumor reactivity after nonmyeloablative lymphodepletion [75]. At the Sheba Medical Center in Israel, administration of TIL led to 10 objective responses out of 20 patients treated, with 2 complete responses [76]. At the Surgery Branch, NCI, the efficacy of TIL therapy is currently being tested for patients with metastatic digestive tract adenocarcinomas.

Gene engineering of peripheral blood T cells is capable of mediating regression of metastatic melanoma and other solid malignancies. The choice of target antigen is critical. Targeting differentiation tumor antigens, such as MART-1, gp100, and CEA, or overexpressed normal proteins such as HER2/neu, may be accompanied with significant toxicities. Targeting the family of cancer-testis antigens, such as NY-ESO, or mutated antigens expressed exclusively by cancer cells, appears promising. Targeting the tumor stroma with a CAR against the vascular endothelial growth factor receptor 2, overexpressed in the tumor vasculature and by some myeloid cells, is an effective strategy in mouse models [77]. Cells with antitumor reactivity could also be used as a “Trojan horse” to deliver other molecules at the tumor site, such as cytokines.