Cancer Immunotherapy

Bacillus Calmette-Guerin

BCG was developed as an attenuated strain of Mycobacterium bovis in 1906, and was being administered as a vaccine to prevent tuberculosis by 1921. BCG was the prototype of nonspecific immune stimulants that were heavily studied as possible cancer therapy in the 1970s. ^26,27 BCG was evaluated as a cancer treatment via intralesional injection, especially for melanoma. ^{28 –30} In addition BCG was evaluated as subcutaneous (s.c.) or intradermal (i.d.) nonspecific immune stimulant for various malignancies. However, randomized trials failed to show a benefit for adding BCG in the adjuvant treatment of breast cancer ^{31 –35} or melanoma. ^{36 –45}

Interest in BCG as an adjuvant for various types of vaccines has persisted for more than 3 decades. ^{46 –50} BCG was the control arm of a large negative trial in which a vaccine consisting of irradiated allogeneic melanoma cells from two cell lines plus BCG proved to be no better than placebo plus BCG in patients with stage III (positive lymph nodes) or stage IV (distant metastases) metastatic melanoma, who were rendered surgically free of disease. ^50,51

A number of trials had demonstrated that intravesical BCG produced responses in patients with superficial bladder cancer, ^{52 –55} including those who had progressed despite prior intravesical therapy. ⁵⁶ Randomized trials from Europe also supported it use. ^{57 –59} BCG (Tice®) was granted FDA approval in 1990 for the intravesical treatment of superficial bladder cancer (in situ and stage I) on the basis of a randomized trial in which BCG was superior to intravesical doxorubicin based on complete response (CR) rates of 70% versus 34% for 131 patients with carcinoma in situ, and a 5-year progression free survival (PFS) of 37% versus 17% for 131 patients with stages Ta, T1. ⁶⁰ Another multicenter trial in 155 patients yielded similar results with a CR of 71% versus 54% for doxorubicin. ⁶¹ BCG continues to be used for this purpose, 30 years after the first reporting of clinical efficacy ⁶² and is still considered the initial treatment of choice for in situ bladder cancer, because no other chemotherapy or agent has proven superior in subsequent randomized trials. ^{63 –68} Recent reports have emphasized the importance of a complete response and long-term maintenance therapy. ^{69 –71}

The precise mechanism by which BCG exerts its antitumor effects is still not well-understood. It is generally believed to result from a nonspecific immune response and accompanying inflammation. After instillation of BCG, it appears that the bacterial organisms are internalized by the bladder epithelium. The resulting mycobacteria glycoprotein complexes apparently induce an inflammatory response involving cellular infiltration and the local release of cytokines that are cytotoxic.

Levamisole

Levamisole (Ergamasol®) is a synthetic phenylimidazothiazole oral antihelminthic that has been used in veterinary practice for several decades. Interest in its use as a cancer agent was spurred by the discovery that it had nonspecific immunostimulatory properties in animals. ^72,73 Levamisole was studied in several relatively small randomized trials in melanoma, ^{74 –77} breast cancer, ^{78 –84} and lung cancer. ^{85 –88} Although some of these trials were considered positive, large confirmatory trials for regulatory approval were never performed in these cancer types.

Although levamisole never gained popularity as treatment in these malignancies, it did achieve some success in the treatment of colorectal cancer. Trials of levamisole alone were not that encouraging, as two randomized trials failed to demonstrate a benefit for levamisole alone, compared to placebo, following surgical resection of node-positive colon cancer. ^89,90 However, randomized trials that combined 5-fluoruracil (5-FU) with levamisole were more encouraging. This included a three-arm British trial that randomly assigned 141 postsurgical patients to observation, 5-FU, or 5-FU plus levamisole. ⁹¹ At 5 years, there was a survival advantage for the group that received levamisole plus 5-FU. ⁹¹ There was also a three-arm, 401-patient U.S. randomized trial that showed an advantage for both levamisole alone (p = 0.05), and 5-FU plus levamisole, compared to observation. ⁹²

Levamisole received FDA approval in 1990 for the adjuvant treatment of colon cancer based on a large randomized, placebo-controlled U.S. trial that demonstrated the superiority of 5-FU plus levamisole compared to 5-FU plus placebo. ⁹³ In this adjuvant trial 1296 patients, who had resected colon cancer that was either locally invasive (n = 318) or metastatic to regional lymph nodes (n = 929), were randomized to observation, or to 5-FU plus levamisole for 1 year, or to levamisole alone. There was no benefit from levamisole alone, but the combination of 5-FU plus levamisole reduced the risk of recurrence and death for patients who had lymph-node metastases. For the node-positive patients (stage III) patients, there was a 40% reduction in recurrence after 6.5 years (p < 0.0001) and a 33% reduction in mortality (p = 0.0007). ⁹⁴ However, there was no evidence of benefit in patients with stage II disease. ⁹⁵ On the basis of this trial, 5-FU plus levamisole became standard therapy for the adjuvant treatment of stage III colon cancer during 1990–1997.

Unfortunately, the pivotal trial that had led to the adoption of levamisole as standard therapy did not have a control arm of 5-FU plus folinic acid (leucovorin), which had also been shown to be more active than 5-FU alone in randomized trials. To clarify this issue, three large U.S. randomized trials were performed. In a four-arm trial that enrolled 891 patients, the combination of 5-FU, leucovorin, and levamisole was superior to 5-FU plus levamisole. ⁹⁶ In a second trial, 2151 patients, who had stage II or III colon cancer, were randomized to receive weekly 5-FU plus leucovorin, or 5-FU plus levamisole, or 5-FU plus leucovorin plus levamisole. ⁹⁷ After more than 7 years of follow-up, the 5-year survival rates were 74% for 5-FU plus leucovorin, 73% for 5-FU plus leucovorin plus levamisole, and 70% for 5-FU plus levamisole. In a third trial 3794 patients were randomized to receive 5-FU plus levamisole for 1 year, or 6 months of low-dose leucovorin plus 5-FU (“Mayo Clinic regimen”), or high-dose leucovorin plus 5-FU (“Roswell Park regimen”), or the Mayo clinic regimen plus levamisole. ⁹⁸ After 10 years of follow-up, there were no differences in survival among the patients in the four arms of the study. Shortly after the results of these trials were first reported in 1997, the 5-FU plus leucovorin regimens replaced the levamisole-containing regimens as standard therapy. During the past decade, four additional randomized trials have been reported that also failed to show a benefit for the addition of levamisole to 5-FU plus folinic acid. ^{99 –102}

Interferon-α

The IFNs are a large family of immune regulatory proteins that are characterized as α, ß, γ, and ω. ¹⁰⁵ IFN-α originally was isolated from leukocytes. Eventually the gene for IFN-α was cloned, and, subsequently, the product was produced by recombinant DNA techniques. The original name was derived from its interference with viral replication. Various IFNs were also found to have wide-ranging immune-stimulating effects and direct cytostatic effects on tumor cells. There were two recombinant DNA products that were extensively studied and eventually approved for widespread use, interferon-α2a (Intron A®) and interferon-α2b (Roferon®). Although studied extensively by s.c., intramuscular (i.m) and intravenous (i.v.) routes, the most popular method of administration is s.c. in doses of 3–20 mIU, given thrice weekly or daily for months to years. IFN-α became the the first biological or immune therapy approved as an anticancer treatment when it received regulatory approval in 1986. There has been clinical experience with IFN-α in virtually every malignancy. Unfortunately, IFN-α has a broad spectrum of toxicities that are dose-related and have included lethal hepatic toxicity. ^{106 –108} In practice, dose-related fatigue and flulike symptoms compromise compliance with planned treatment dosing and schedules, but it is much better-tolerated when administered late in the day in combination with anti-inflammatory agents.

Hairy-cell leukemia. IFN-α was the first popular “biological response modifier” and, as such, was extensively studied in virtually every malignancy. ^109,110 In 1986, IFN-α was approved by the FDA for the treatment of hairy-cell leukemia based on a response rate (RR) of 70%–75% that proved to be quite durable. ^{111 –117} By the early 1990s, INF-α had been displaced by pentostatin as the treatment of choice for this uncommon malignancy because of its activity in patients whose disease was refractory to IFN-α ¹¹⁸ and pentostatin's superiority to IFN-α in a randomized trial. ¹¹⁹ Both pentostatin and cladribine produced a higher RR with less toxicity and were associated with even longer durations of remission. However, it is noteworthy that IFN-α can still be useful in treating hairy-cell leukemia after it has become refractory to purine analogs.

Chronic myelogenous leukemia. IFN-α proved to be active in chronic myelogenous leukemia (CML) with a clinical RR of ∼80%. ^120,121 In addition to durable clinical responses, high-grade cytogenetic responses were confirmed as well. ^122,123 In a landmark Italian trial that enrolled 322 CML patients during 1986–1988, IFN-α was shown to be superior to hydroxyurea or busulfan chemotherapy in the treatment of newly diagnosed chronic-phase CML. ¹²⁴ A German trial that randomized 513 CML patients to IFN-α or hydroxyurea or busulfan confirmed the superiority of IFN-α over busulfan, and IFN-α was as effective as hydroxyurea. ¹²⁵ A Japanese randomized trial involving 159 patients also confirmed a superior survival for patients treated with IFN-α rather versus busulfan. ¹²⁶ Except for patients who were candidates for allogeneic transplants, for more than a decade, IFN-α was the treatment of choice for CML. ¹²⁷ In 1997, IFN-α plus cytarabine was proven to superior to IFN-α alone, but, within a few years, this combination was displaced by the tyrosine kinase inhibitor imatinib. ^128,129

Melanoma: Metastatic. As a single agent IFN-α was associated with objective RRs ranging from 5% to 25% in patients with metastatic melanoma, who were treated via a variety of different routes, doses, and schedules. ^130,131 An optimal route, dose, and schedule were never determined, and there were never any randomized trials of IFN-α versus the standard chemotherapy agent dacarbazine. A number of randomized trials evaluated whether IFN-α provided any benefit when added to chemotherapy. One 64-patient randomized trial suggested a higher RR and better survival for the combination of dacarbazine and INF-α, compared to dacarbazine alone, ¹³² but subsequent randomized trials failed to confirm such an advantage. ^{133 –136} There were no randomized trials of combination chemotherapy ± IFN-α, but a multicenter phase II trial confirmed added toxicity in association with an RR and survival that appeared to be no better than the same chemotherapy alone, when IFN-α was added to carmustine (BCNU), cisplatin, dacarbazine, and tamoxifen. ¹³⁷

A randomized trial failed to confirm an advantage for combining IFN-α with high dose IL-2, ¹³⁸ or with cisplatin and IL-2. ¹³⁹ Several randomized trials examined the addition of these same two cytokines with combinations of chemotherapy that included cisplatin, dacarbazine with tamoxifen, ¹⁴⁰ or BCNU, cisplatin, and dacabazine alone, ¹⁴¹ or with tamoxifen. ^142,143 No trial demonstrated a benefit for the addition of the two cytokines. A 183-patient, single center, randomized trial did find a higher RR and survival advantage (p = 0.06) for the addition of i.v. IL-2 and s.c. IFN-α in combination with cisplatin, vinblastine, and dacarbazine (DTIC). ¹⁴⁴ However, a subsequent multicenter trial with the same treatment protocol failed to confirm such benefit, ¹⁴⁵ and another trial using the same agents, but with a different dose and schedule of IL-2 and IFN-α, also showed no benefit for the addition of the cytokines, compared to the combination chemotherapy. ¹⁴⁶ The use of IFN-α in the treatment of metastatic melanoma has decreased over the past several years.

Melanoma: Adjuvant. The major use of IFN-α for more than a decade has been as adjuvant treatment after resection of melanoma that is associated with a high risk of recurrence. In 1995, a negative trial of adjuvant i.m. IFN-α was published, ¹⁴⁷ but the following year, the positive results of European Cooperative Oncology Group (ECOG) 1684 appeared, which demonstrated prolonged progression-free survival (PFS) and overall survival (OS), compared to observation alone. ¹⁴⁸ This landmark trial was conducted between 1985 and 1990 in patients with deep, locally advanced, lymph–node positive, or regional lymph-node recurrence of melanoma. In fact, the largest subset of patients entered were from the last category rather than having newly diagnosed stage II or III melanoma. The induction treatment consisted of 20 mIU/m2 for 5 of 7 days during weeks 1 through 4, followed by 10 mIU/m² s.c., thrice weekly for 48 weeks. Only 25% of patients were able to complete the planned 1 year of treatment, with most stopping therapy because of toxicity, especially fatigue. Despite this, quality-of-life (QoL) studies showed that therapeutic benefit outweighed the risks. ¹⁴⁹ In the intervening years, it was determined that evening injections and judicious use of anti-inflammatory agents increase the 1-year compliance rate to 75%.

The ECOG 1684 trial has been updated many times. After capturing more complete outcome data on patients whose data had originally been censored, it was finally concluded that IFN-α conveyed a PFS benefit, but lacked a statistically significant benefit for OS. Another trial confirmed the superiority of this regimen, compared to a lower-dose IFN-α regimen on the basis of superior PFS. ¹⁵⁰ A third trial confirmed that this regimen was associated with a longer time to progression (TTP) and OS, compared to a disialoganglioside vaccine. ¹⁵¹ A pooled analysis of 713 patients enrolled in these trials strongly supported evidence for prolonged PFS, but showed no difference in OS. ¹⁵² A subsequent analysis of these trials concluded there was a 15% reduction in death after 2 years (p = 0.03) and the researchers endorsed this high-dose regimen as the standard adjuvant therapy. ¹⁵³

A number of specific laboratory immunologic parameters were demonstrated to have changed during IFN-α therapy, but none were predictive of survival. ¹⁵⁴ However, subsequent analyses showed that the patients treated with IFN-α who had the best survival were those who developed antithyroid, antinuclear, antiDNA, and/or anticardiolipin autoantibodies. ¹⁵⁵

In recent years, some trials have suggested that 1 month of high-dose i.v. IFN-α may be as effective as a full year of treatment. In an effort to decrease toxicity, a trial was conducted comparing 1 month of a somewhat lower i.v. IFN-α therapy (15 mIU/m² for 5 of 7 days, during weeks 1 through 4) to a year-long program that included the lower i.v. induction dose, and another study arm that consisted of the same high-dose i.v. induction and 11 months of s.c. IFN-α as used in ECOG 1684. ¹⁵⁶ There was no significant difference in disease-free survival (DFS) or OS.

Because of the toxicity of the ECOG 1684 IFN-α regimen, numerous lower-dose s.c. regimens have been tested, especially in Europe. Most trials utilized 3 mIU s.c. thrice weekly. None of these regimens were superior to observation in patients with resected lymph-node metastases. Three randomized trials utilizing low-dose s.c. IFN-α in patients with node-negative melanoma, demonstrated a prolonged PFS, but no increase in OS, ^{157 –159} while a fourth trial found no benefit in either metric. ¹⁶⁰ Low-dose s.c. regimens showed no evidence of benefit in two randomized trials in patients with clinically node-positive disease, ^161,162 or in trials that included patients with deep primary lesions (3 or 4 mm greater) and/or locoregional metastases. ^63,164 Similarly, in a three-arm trial of 1388 patients whose melanoma was either ≥4 mm in depth and/or associated with positive regional nodes, intermediate doses of thrice weekly adjuvant IFN-α at 10 mIU for 1 year or 5 mIU for 2 years, conveyed no benefit, compared to observation. ¹⁶⁵ In a trial that randomized 650 node-positive patients to induction with 10 mIU/m² of i.v. IFN-α on 5 of 7 days for 2 weeks followed by s.c. IFN-α, or low-dose s.c. IFN-α for 2 years, there was no difference in TTP or OS. ¹⁶⁶ In contrast to these trials, adjuvant therapy with s.c. pegylated-IFN-α (PEG-IFA-α), a product designed to sustain serum levels longer with fewer injections, did provide a DFS benefit in node-positive patients with an 18% reduction in relapse at a median follow-up of a little over 3 years. ¹⁶⁷ QoL analysis confirmed relative deterioration in the IFN-α arm in terms of social and role function as well appetite, fatigue, and dyspnea. ¹⁶⁸ Appearance of autoimmune antibodies was not predictive of benefit from PEG-IFN-α. ¹⁶⁹

Several randomized trials added other agents in an effort to improve on IFN-α alone as an adjuvant therapy. In a placebo-controlled trial of more than 400 node-negative stage II patients (>3 mm deep), there was no advantage for the addition of isoretinoin. ¹⁷⁰ Administration of a vaccine derived from allogeneic tumor cell lysate combined with low-dose s.c. IFN-α produced survival results similar to those achieved with the ECOG 1684 high-dose regimen and was less toxic. ¹⁷¹ In a three-arm trial of low-dose IFN-α, observation, or IFN-α plus DTIC in 441 patients with node-positive disease, there was a survival advantage for IFN-α alone compared to observation, but this benefit was lost in the arm that contained DTIC, suggesting that chemotherapy may have suppressed any beneficial immune response. ¹⁷² In a trial that was stopped early because of slow accrual, there were no differences among the arms of a trial in which a biochemotherapy arm that included both IL-2 and IFN-α was compared to high- and low-dose regimens of IFN-α. ¹⁷³

Renal cell cancer. For many years IFN-α has been considered a standard agent for the treatment of renal cell carcinoma based on published RR in the range of 10%–20% in phase II trials. ^{174 –182} An analysis of 463 patients treated with IFN-α at Memorial Sloan Kettering in six different prospective trials revealed a median PFS of 4.7 months and a median OS of 13 months. ¹⁸³ Two randomized trials compared single-agent IFN-α to medroxyprogesterone and confirmed a higher RR and better survival for patients who received IFN-α. ^184,185 IFN-α has been combined with a number of other agents, and some randomized trials have been conducted to determine whether there is an advantage for the combinations. In two randomized trials involving IFN-α plus vinblastine, the combination was superior to vinblastine alone ¹⁸⁶ and to medroxyprogesterone. ¹⁸⁷ In three randomized trials involving IFN-α and cis-retinoic acid (CRA), one trial detected an improved PFS and OS for the combination over IFN-α alone, ¹⁸⁸ but the other two trials found no difference in outcome. ^189,190 The combination of IFN-α, low-dose IL-2, and 5- FU, was no better than IFN-α alone. ¹⁹¹ IFN-α plus s.c. IL-2, with or without 5-FU, was superior to IFN-α and vinblastine in patients with advanced renal cell cancer. ¹⁹² The combination of IFN-α, IL-2, and 5-FU failed to provide a survival advantage, compared to observation alone in the adjuvant treatment of high-risk, resected kidney cancer. ¹⁹³ More recently, the addition of bevacizumab to IFN-α was found to be superior to IFN-α alone in two large randomized trials. ^194,195 IFN-α has been used as the control arm to establish the superiority of other agents for the treatment of renal cell cancer. Temsirolimus with or without INF-α was superior to IFN-α alone, but the combination was no better than temsirolimus alone in a trial of 626 patients with poor-prognosis metastatic renal cell cancer. ¹⁹⁶

B-cell lymphoma. Early phase II trials suggested that IFN-α has activity as a single agent in the treatment of indolent B-cell lymphoma. In the pre-rituximab era, five randomized trials in indolent B-cell lymphoma, from five different countries, established that the addition of IFN-α to induction chemotherapy that did not include an anthracycline had no significant impact on RR or survival, but maintenance IFN-α was associated with a modest increased TTP in most of the trials. ^{197 –201} Two multicenter randomized trials showed that the addition of IFN-α enhanced RR, PFS, and OS when combined with chemotherapy that included doxorubicin in the treatment of more aggressive lymphomas. ^{202 –205} However, a third trial failed to confirm any benefit from this approach. ²⁰⁶ A large trial, in which follicular lymphoma patients were treated with ProMACE-CytoBOM followed by randomization to IFN-α maintenance therapy, showed no difference in any of the response or survival parameters^{. 207} Eventually, a meta-analysis of these trials, most of which began in the 1980s, concluded that IFN-α did prolong survival and remission duration in patients with follicular lymphoma but only in the context of relatively intense induction therapy and when a dose of at least 5 mIU units per dose or at least 36 MIU per month was administered. ²⁰⁸ The introduction of the highly effective anti-CD20 monoclonal antibody rituximab in 1997 resulted in an immediate decrease in the use of IFN-α in the treatment of B-cell lymphoma, although this use persisted for maintenance therapy for some time in lymphoma clinical trials, based on the data cited above.

Cutaneous T-cell lymphoma. As a single agent, IFN-α had produced RR as high as 50% and CR rates of 20% in various studies in stage I and II cutaneous T-cell lymphoma (CTLC) patients (placque phase or mycosis fungoides). ²⁰⁹ In mycosis fugoides IFN-α is widely used in combination with photochemotherapy (psoralen plus ultraviolet A; PUVA), a combination that has been shown to be superior to IFN-α plus acitretin. ²¹⁰ IFN-α is considered useful in more advanced stages of the disease.

Superficial bladder cancer. IFN-α is sometimes used in the intravesical treatment of bladder cancer, based on CR rates of 20% to 45% in patients with residual superficial disease ^211,212 and a small reduction in the 1-year recurrence rate. ²¹³ Direct comparison in a randomized trial showed that IFN-α was not as effective as BCG in preventing recurrence. ⁶⁶ However, the combination of IFN-α and BCG is often effective in patients who have progressed after prior treatment with BCG. ^214,215 IFN-α can be combined with chemotherapy agents as well, and the combination of IFN-α and mitomycin C was more effective than either agent alone. ²¹⁶ Similarly, the combination of IFN-α and epirubicin was more effective than epirubicin alone. ²¹⁷

Gastrointestinal malignancies. Although IFN-α never demonstrated much single-agent activity in gastrointestinal (GI) malignancies, for many years there was great enthusiasm for the combinations of IFN-α and 5FU-based therapie, based on in vitro evidence of synergy and based on encouraging results from small single-institution studies. ²¹⁸ Eventually, however, a series of randomized trials established that adding IFN-α to chemotherapy only increased toxicity without adding therapeutic benefit in colorectal cancer. ^{219 –232} A meta-analysis of 1764 patients enrolled in 12 randomized trials concluded that IFN-α does not increase the efficacy of 5FU or 5FU + leocovorin, and that 5FU + IFN-α is significantly inferior to 5FU + LV, for patients with advanced colorectal cancer. ²³³ Randomized trials also confirmed lack of benefit in the adjuvant setting in either colon or rectal cancer. ^{234 –237}

Interleukin-2

IL-2 (Proleukin®) was the first biological approved for the treatment of cancer, based entirely on immune modulatory effects, in as much as IFN-α also has direct antitumor cytostatic effects in addition to its immune-modulating effects. ²³⁸ In the United States IL-2 gained regulatory approval for marketing in renal cell cancer (1992) and melanoma (1997). IL-2 has no direct antitumor effects but induces cytotoxic natural-killer (NK) and T-lymphocytes. ²³⁹

IL-2 was originally isolated as T-cell growth based on activity from secretions in media by antigen-activated helper T-cells. ²⁴⁰ Cloning of the IL-2 gene made possible the large-scale production of IL-2 by recombinant DNA techniques for clinical use. The original clinical interest in IL-2 was focused on its role in the ex vivo culture of T-lymphocytes, especially for possible clinical application of cytotoxic T-lymphocytes (CTLs). ^241,242 This is why most early trials focused on IL-2 in combination with adoptive cell-therapy products known as lymphokine-activated killer (LAK) cells, or tumor-infiltrating lymphocytes (TILs). The most encouraging results were in melanoma and renal cell cancer, which were generally considered to be immunoresponsive tumors, but some activity was seen in virtually every tumor type. ^{243 –248}

The most popular IL-2 regimens were the high-dose bolus regimen, developed at the National Cancer Institute (NCI), and the 5-day continuous-infusion approach developed by West et al., ²⁴⁶ which proved to be more practical in the community setting. ²⁴⁹ The only randomized trial that ever compared these regimens found no difference in RR or PFS in patients with renal cell cancer. ²⁵⁰ Originally both approaches utilized 5-days of IL-2 to activate T-lymphocytes in vivo, followed by collection of lymphocytes by leukapheresis for additional stimulation with IL-2 in vitro, and then an additional 4–5 days of IL-2 in association with infusion of LAK cells. Long-term survival of patients has been documented for both the high-dose bolus ^{251 –253} and the high-dose continuous-infusion regimens. ^{254 –257} Although IL-2 was explored in other malignancies—especially lymphoma, lung cancer, and colorectal cancer—benefit was never established in cancers other than renal cell and melanoma. ^238,258,259

Experience showed that IL-2 had activity with or without LAK cells, whether given as a high-dose bolus (NCI) or on the continuous-infusion schedule. ^245,260,261 Small randomized trials of high-dose IL-2 with or without LAK cells failed to show a dramatic benefit for the adoptive cell-therapy component in either renal cell cancer or melanoma. ^{262 –264} Based on these observations, and the complexity of adoptive cell-therapy production, commercial development focused on trials conducted with IL-2 alone, which eventually led to regulatory approval of the continuous-infusion regimen in Europe, and the high-dose bolus regimen in the United States.

When reviewing the older IL-2 literature, it is important to note that 1 mg of IL-2 is the equivalent of 3 million units (MU) of Cetus IL-2, which is the equivalent of 18 million international units (mIU) and about 6 MU of Hoffman-LaRoche IL-2. Also, what was originally reported as 720,000 units/kg/dose of bolus IL-2 was actually 600,000 international units (IU). Today, the high-dose bolus IL-2 dose and schedule is 14 doses at 600,000 units/kg over 5 days. The high-dose continuous-infusion dose and schedule is 18 mIU per square meter of body surface (m²) per day for 5 days. Thus a 5′ 9″, 85 kg man (2.0 m²) would have received 714 mIU over 5 days by the bolus schedule, and 180 mIU by the continuous-infusion schedule. The trials utilized the same dosing and scheduling applied for IL-2 plus LAK, and simply inserted a 5-day rest period between the two 5-day treatments that constituted one treatment cycle. Thus, for one treatment cycle, the high-dose schedule was planned for 1428 mIU over 15 days while the continuous i.v. infusion schedule was planned for 360 mIU over 15 days, or ∼25% of the high-dose bolus dosing.

The trials of IL-2 alone established the efficacy and toxicity of the agent as a monotherapy for renal cell cancer, ^{265 –268} and melanoma. ^269,270 Based on the compilation of results from these phase II trials, IL-2 was granted a marketing indication for renal cell cancer in 1992 and for melanoma in 1997. In renal cell cancer a RR of 15% was observed in 255 patients enrolled in seven different phase II trials of high-dose IL-2, but the 7% of all treated patients who experienced a CR had a PFS of greater than 5 years. In melanoma, a 16% response rate was observed in 270 patients from eight phase II trials, but the 6% of all treated patients who experienced a CR had a median survival of >5 years.

One of the greatest clinical challenges for IL-2 has always been the severe side-effects. ²⁷¹ These included severe flulike symptoms, fever, chills, nausea, vomiting, diarrhea, cardiac arrythmias, hepatic enzyme elevation, exfoliative dermatitis, and extravasation of fluid that was associated with renal insufficiency, encephalopathy, pulmonary edema, and hypotension, and sometimes resulted in death. For this reason, the high-dose IL-2 treatment was typically carried out in intensive care units (ICUs), while the continuous-infusion approach proved practical for inpatient cancer units. Higher doses of IL-2 are required for maximum benefit, but, unfortunately, this is associated with significant toxicity that requires hospitalization, and most practicing oncologists are not skilled in high-dose IL-2 administration. ²⁵⁹

There were many claims of efficacious lower-dose outpatient regimens both by i.v. and s.c. routes, but most of these represented the experiences of single institutions and therefore were subject to unintentional patient selection bias. Small multicenter phase II trials suggest that a hybrid high-dose bolus and 3-day continuous-infusion schedule is less toxic and less costly, and as efficacious as the 5-day continuous-infusion schedule, ^{272 –274} but randomized comparative trials have not been performed. In patients with renal cell cancer, a randomized trial did confirm that higher doses of bolus IL-2 are associated with superior RR compared to lower-dose bolus regimens or s.c. regimens, and survival trends also favored the highest bolus dose. ²⁷⁵ In contrast, a French randomized trial discerned no difference between continuous infusion i.v. and lower-dose s.c. IL-2 when used in combination with s.c. IFN-α in renal cell cancer patients. ²⁷⁶ In addition to clinical toxicity, another recently recognized limitation of IL-2 is that it not only stimulates cytotoxic NK and T-cells but also regulatory T-cells that may counter some of the antitumor effects of the cytotoxic cells. ²⁷⁷

Renal cell cancer. Inpatient high dose IL-2 alone remains a standard treatment for patients with advanced renal cell cancer and has been associated with long-term survival and cure, but only a small percentage of patients actually benefit from such therapy. ²⁷⁸ In an effort to improve the results of high-dose IL-2 alone, IFN-α has been combined with high-dose bolus IL-2, ^279,280 with high-dose continuous-infusion IL-2, ^281,282 and with a hybrid high-dose bolus and continuous-infusion IL-2 schedule. ²⁷⁴ Both high-dose bolus IL-2 and high-dose continuous-infusion IL-2 have been combined with mono-chemotherapy, or combination chemotherapy, but none of these approaches has proven superior to high-dose IL-2 alone.

Other trials have focused on giving lower doses of IL-2, especially via the s.c route in the outpatient setting. ^{283 –287} Lower-dose i.v. bolus schedules have also been explored. ²⁸⁸ Eventually a randomized trial established that higher doses of bolus IL-2 are associated with superior RR, compared to lower-dose i.v. bolus regimens or s.c. regimens, and survival trends also favored the highest bolus dose. ²⁷⁵

Low-dose s.c. regimens were extensively evaluated in combination with IFN-α in a variety of doses and schedules. ^{289 –295} In addition IFN-α was combined with a lower-dose bolus regimen of IL-2. ²⁹⁶ In a small randomized trial, low response rates were observed with both s.c. IL-2 alone and with IFN-α in 60 patients with metastatic renal cell cancer. ²⁹⁷ A French randomized trial detected no difference between continuous infusion i.v. and lower dose s.c. IL-2, when used in combination with s.c. IFN-α in 155 renal cell cancer patients. ²⁷⁶ In a randomized trial in which 183 metastatic renal cell cancer patients received s.c. IL-2 and s.c. IFN until disease progression, but one arm continued treatment even in the face of disease progression, there was a 14-month median survival in each arm. ²⁹⁸ There was one trial in which 425 patients with metastatic renal cell carcinoma were randomly assigned to receive a continuous infusion of IL-2, s.c. IL-2, or both. RR and event-free survival (EFS) were both better in the combination arm, but there was no difference in survival. ²⁹⁹

Lower-dose IL-2 was also combined with mono-chemotherapy, ^300,301 but most studies focused on the combination of 5-FU, IL-2, and IFN-α ^{302 –307} RRs as high as 30% to 50% were reported from single institutions, but larger multicenter trials resulted in RRs ranging from 0% to 18%. In a multicenter trial that combined these three agents with cis-retinoic acid, the objective RR was only 6%. ³⁰⁸ Other agents added to IL-2 and IFN-α have included cyclophosphamide, ³⁰⁹ vinblastine, ³¹⁰ and gemcitabine. ³¹¹

IL-2 has also been combined with other biologicals besides IFN-α, including IFN-γ, ³¹² anti-CD3 monoclonal antibody, ^313,314 levamisole, ³¹⁵ GM-CSF, ^316,317 GM-CSF plus IFN-α, ³¹⁸ thalidomide, ^319,320 bryostatin, ³²¹ and the histamine blocker famotidine. ^322,323 Most of these approaches were abandoned after small exploratory trials, and none has ever proven superior to high-dose IL-2 alone with respect to long-term survival benefit. ²⁹³ Very few randomized trials were ever performed with these regimens.

Melanoma. Inpatient high-dose IL-2 alone remains a standard treatment for patients with metastatic melanoma, and this approach has been associated with long-term survival and cure. Trials exploring lower doses of i.v. bolus IL-2 alone or lower dose continuous-infusion IL-2 alone have yielded low RRs and poor disease control. ^288,324,325 In 26 patients with a median age of 45, for whom biochemotherapy utilizing lower doses of IL-2 had failed, subsequent treatment with high-dose bolus IL-2 produced a 19% response with four CRs that lasted more than 2 years. ³²⁶

In an effort to improve the results of high-dose IL-2 regimens, other agents have been combined with this therapy. The addition of IFN-α alone resulted in high RRs in single-institution trials ²⁷⁹ but proved to be no better in multicenter randomized trials than high-dose bolus IL-2, ¹³⁸ or high-dose continuous-infusion IL-2. ²⁸¹ DTIC has been a standard agent in the treatment of melanoma for 3 decades and was a logical choice to combine with IL-2. Several small phase II trials of sequential therapy yielded RRs of only 13%–25%, which did not appear to be better than either agent used alone. ^{327 –330} Other trials combined lower doses of IL-2 with mono-chemotherapy. Response rates of 15%–25% were reported for low-dose cyclophosphamide combined with a low-dose bolus of IL-2. ^300,301 Similar results were obtained with an outpatient bolus IL-2 regimen and dacarbazine. ³³¹ The highest RRs were reported from a multicenter trial that combined cisplatin, dacarbazine, and tamoxifen with a high-dose bolus of IL-2, which resulted in an RR of 42%. ³³²

Other trials focused on combining lower doses of i.v. or s.c IL-2 with IFN-α. Two successive German trials utilizing high doses of s.c. IL-2 resulted in RRs of 18% and 41%. ³³³ A trial of low-dose continuous-infusion IL-2 plus s.c. IFN-α produced an RR of 16% in 57 patients, ³³⁴ and a U.S. trial of a low-dose continuous IL-2 with s.c. IFN-α resulted in a response rate of only 8%. ³³⁵ One trial utilizing a low-dose bolus of IL-2³³⁶ and another trial using decrescendo continuous-infusion IL-2 over 72 hours ³³⁷ reported no ORs in patients with measurable metastatic melanoma.

Because of the early reports suggesting that combinations of IL-2 and IFN-α were better than either agent alone, the most popular approaches combined these two biological agents with one or more chemotherapy agents, usually in a sequential or alternating schedule. Single institutional studies using these strategies yielded RRs of 35%–55% with median survival rates of 10 months to 1 year. ^{338 –346} A retrospective analysis of patients treated outside a protocol setting wth cisplatin, vinblastine, and dacarbazine (CVD) plus s.c. IFN-α and two different i.v. doses of IL-2, given over 4 days, determined RRs of 5/29 (30%) for the lower dose of IL-2 and 15/37 (41%) for a higher dose. ³⁴⁷ RRs and survival rates were somewhat lower in higher-risk patients and in multicenter trials. In a German trial of patients who were considered poor risks for intensive IL-2 because of very advanced disease, 24% of 21 patients had a PR in a trial combining dacarbazine, cisplatin, and decrescendo continous i.v. IL-2 and s.c. IFN-α. ³⁴⁸ Successive trials of s.c. IL-2 and s.c. IFN-α and cisplatin with or without cimetidine produced an RR of 27% and a median survival of 10.2 months for 82 patients. ³⁴⁹ A community-based multicenter trial of BCNU (carmustine), cisplatin, dacarbazine and taxmoxifen (BCDT) interdigitated with low-dose s.c. IL-2 and IFN-α resulted in a 38% RR and 42% of the subjects were alive at 1 year. ³⁵⁰ In two successive multicenter phase II trials, dacabazine, cisplatin and IFN-α were interdigitated with either 4 days of outpatient i.v. bolus. IL-2, or 4 days of low-dose s.c. IL-2. ³⁵¹ RRs were 17/44 (36%) for the i.v. regimen and 6/36 (17%) for the lower-dose s.c. regimen, and the median survival rates were 10.7 and 7.3 months, respectively. The combination of cisplatin, vinblastine, dacarbazine and i.v. IL-2 plus IFN-α was associated with RRs of 60% in a single-institution trial ³⁵² and 48% in a multicenter trial, ³⁵³ although the schedules and doses of biotherapy differed.

Several randomized trials have addressed different components of these biochemotherapy regimens using a single chemotherapy agent. A randomized trial showed that the addition of cisplatin to IL-2 plus IFN-α resulted in a higher RR and better PFS, but both groups had a median survival of only 9 months, with no difference in survival curves. ³⁵⁴ In a randomized trial that enrolled 117 patients, there was no advantage for the addition of IFN-α to the combination of cisplatin and high-dose continuous infusion IL-2. ³⁵⁵ The slightly higher RRs (21% versus 16%), and median survival (10.9 versus 10.4 months) associated with the addition of IFN-α were not statistically significant. In another randomized trial 290 patients were randomized to dacarbazine plus IFN-α with or without a hybrid of low-dose bolus IL-2 and s.c. IL-2. ³⁵⁶ There was no difference in RRs (16% and 18%) and median survival was 11 months in both arms. These randomized trials suggested that there was no advantage to chemotherapy plus IL-2 and IFN-α, compared to the biotherapy doublet alone or a biochemotherapy doublet with either immunotherapeutic.

Randomized trials of combination chemotherapy, as a component of biochemotherapy, have yielded similar results. Three randomized trials addressed the issue of whether the addition of immunotherapy provided a benefit over BDCT combination chemotherapy. In a British randomized trial that enrolled 65 patients and utilized s.c IL-2 and IFN-α in combination, the RRs were 23% for the combination arm and 27% for chemotherapy alone, with a remarkably poor median survival of 5.5 months. ¹⁴² In a U.S. trial that enrolled 102 patients, the addition of IFN-α plus a high-dose bolus IL-2 after combination chemotherapy was associated with a higher response rate (44% versus 27%), but survival was better in the chemotherapy-alone group (median 11 months versus 16 months). ¹⁴⁰ However, in that trial, patients who were randomized to chemotherapy alone typically received high dose IL-2 at the time of relapse. This suggests that in good-risk patients, single modality therapy may be appropriate initially with the other modality reserved for relapse. None of these approaches has ever been directly compared to high-dose bolus IL-2 alone. A third randomized trial examined whether the addition of low dose s.c. IL-2 and s.c. IFN-α improved the outcome for patients receiving dacarbazine and cisplatin with or without carmustine. ¹⁴¹ The 89 patients randomized to chemotherapy had an RR of 20% and a median survival of 9.5 months, which was not different than the 25% response rate and 11-month survival for the 87 patients who received immunotherapy.

Randomized trials have also addressed the issue of whether IL-2 based biotherapy adds to the chemotherapy combination of dacarbazine, cisplatin, and vindesine or vinblastine (CVD). A 92-patient 3-arm trial randomized patients to receive dacarbazine and IL-2, or CVD (with vindesine) and IL-2, or CVD, with IFN-α and tamoxifen given in all three arms. ³⁵⁷ The two arms that used low-dose s.c. IL-2 were associated with a higher RR (35% and 37% versus 13%), but there was no advantage for combination chemotherapy. In a 151-patient, 2-arm trial of CVD ± s.c. IL-2 and s.c. IFN-α, there was a slight increase in RR (33% versus 21%) but no improvement in PFS survival or OS (11–12 months). ¹⁴⁵ In an 89-patient randomized trial comparing biochemotherapy with dacarbazine, cisplatin, IL-2 and IFN-α to two cycles of dacarbazine alone, followed by the same biochemotherapy, there was better initial disease control for the immediate biochemotherapy, but overall survival was similar (10.5 versus 9.5 months). ³⁵⁸

In a 190 patient randomized trial CVD (with vinblastine) was given with or without inpatient continuous infusion IL-2 with s.c. IFN, which immediately followed each cycle of chemotherapy. ¹⁴⁴ Results favored the biochemotherapy arm (91 evaluable patients) over the chemotherapy-alone arm (92 patients) in terms of objective RR (48% versus 25%, p = 0.001), TTP (4.9 versus 2.4 months, p = 0.008), and OS (11.9 versus 9.2 months, p = 0.059). A randomized trial with 395 evaluable patients tested the same biochemotherapy using a different schedule for the biotherapy component. ¹⁴⁶ There was a higher objective RR for biochemotherapy (20% versus 14%, p = 0.14), and longer PFS (4.8 versus 2.9 months, p = 0.015), but no difference in OS (9.0 versus 8.7 months). In a 363-patient randomized trial there was no benefit from the addition of decrescendo i.v. IL-2 and s.c. IFN-α to the chemotherapy doublet of dacarbazine and cisplatin in terms of RR (21% versus 231%), PFS (3.9 versus 3.0 months) and OS (median 9 months in both arms). ³⁵⁹

In the adjuvant setting, no benefit has been documented for IL-2–based therapy. In a randomized trial conducted in patients with node-negative melanoma, there was no benefit for the addition of IL-2 and IFN-α in terms of DFS or OS. ¹⁶⁰ In a trial that assessed 650 node-positive patients randomized to induction with 10 mIU/m² of i.v. IFN-α for 5 of 7 days for 2 weeks followed by s.c. IFN-α, or low-dose s.c. IFN for 2 years, there was no difference in DFS or OS. ¹⁶⁶

Other trials involving melanoma patients, combined IL-2, with or without chemotherapy, with other biologicals, such as an anti-CD3 monoclonal antibody, ³¹⁴ R24, an antimelanoma antibody that reacts to disialoganglioside GD3, ³⁶⁰ levamisole, ³⁶¹ histamine dihydrochloride, ^{362 –364} the pineal hormone melatonin, ³⁶⁵ the gp100 peptide antigen, ³⁶⁶ famotidine, ³⁶⁷ IFN-α plus GM-CSF, ³⁶⁸ and lymphodepleting chemotherapy plus GM-CSF. ³⁶⁹ Only a few randomized trials have been carried out to test these approaches. Histamine plus IL-2 was not superior to IL-2 alone in patients with stage IV melanoma, although issues were raised regarding potential benefit compared in a subset of patients with hepatic metastases. ³⁶³ Another trial found that the combination of IL-2, IFN-α, and histamine dichloride was no better than dacarbazine. ³⁶⁴ The most encouraging randomized trial to date is the initial report describing a possible survival benefit when gp100 was combined with high-dose bolus IL-2 compared to IL-2 alone. ³⁷⁰

Today, IL-2 delivered by the higher-dose intensive regimens as a single agent is still considered an active agent in melanoma, and the only approach for which long-term disease control and survival have been documented. Unfortunately, this therapy is so toxic that it requires careful patient selection, and can only be safely infused by physicians and nursing staffs experienced in its administration. As in renal cell cancer, for patients with metastatic melanoma, most of the promising results from single-institution, small phase II studies were not confirmed in multicenter trials and/or randomized trials. Many of the combination regimens appear to have higher RRs, and perhaps longer PFS, but long-term OS benefits have not been documented. Because high-dose IL-2 regimens are only appropriate for younger, healthier patients, in practice, more melanoma patients are treated with combination regimens that use lower doses of IL-2.

Granulocyte-macrophage colony-stimulating factor (GM-CSF)

GM-CSF (Leukine®) is an important cytokine that has profound effects on the differentiation and actions of myeloid cells. ³⁷¹ It has not been approved as a monotherapy for cancer treatment, but is one of several hematopoeitic stimulating factors that have been approved for clinical use because of their positive effects on recovery of hematopoietic cells. GM-CSF was approved in 1992 specifically to expedite the recovery of granulocytes after cytotoxic therapy. ^372,373 However, because of GM-CSF's effects on monocytes, macrophages, and dendritic cells, it is of great interest for immunotherapy. ^{374 –376} GM-CSF is being widely studied as an adjuvant with vaccines, either by co-administration or by genetic modification of the vaccine product. ³⁷⁷

As a monotherapy, one phase II trial found that patients with metastatic melanoma who had regionally advanced or limited metastatic disease that had been surgically resected, and then received adjuvant GM-CSF, had a better DFS and OS compared to historical controls that were not truly representative of modern patients. ³⁷⁸ In 98 patients treated for 3 years with GM-CSF following surgical resection of deep melanomas (T4), or regional lymph-node metastases, or distant metastatic disease, the melanoma-specific 5-year survival rate was 60%. ³⁷⁹ To date, randomized trials have yet to produce results to justify a marketing indication for GM-CSF as an anticancer agent. Between 1999 and 2006, 815 patients with surgically resected stage III or stage IV melanoma, and who were HLA-2 negative, and therefore ineligible for a gp100 vaccine trial, were randomized to GM-CSF or placebo. ³⁸⁰ The results suggest that the adjunctive use of GM-CSF may convey an advantage in terms of DFS in such patients, but not OS.

Natural-killer cells and lymphokine-activated killer cells

NK cells are large granular lymphocytes ³⁸³ and small agranular lymphocytes, ³⁸⁴ that are inherently cytotoxic against tumor cells in the absence of immunization or activation. ^{385 –388} They constitute ∼ 10% of circulating lymphocytes. NK were first characterized as “null cells” that did not express antigens that were used to identify B- and T-cells. ³⁸⁹ Subsequently, a subset characterized as NK T-cells (NKT) was identified. ³⁹⁰ NK cells are cytotoxic against K562 cells, virally infected autologous cells, and autologous tumor cells that lack expression of major histocompatibility complex (MHC) class I antigens or B7 molecules. NK cells can also affect ADCC via the CD16 FcγRIII that binds to the Fc portion of antibody-coated cells. The majority of NK cells are phenotypically CD2-, CD3-, CD4-, CD8-, CD19-, CD20-, CD16+, CD25+, and CD56+; are directly cytotoxic; and can serve as effector cells in ADCC. Although unstimulated NK cells can recognize and kill tumor cells in vitro, there have been no large-scale clinical trials of unstimulated NK cells. Instead, such cells have typically been stimulated with interleukin-2 (IL-2) and infused into patients with additional IL-2.

Much of the original interest in the cytokine IL-2 was because of enhanced cytotoxic effects of NK cells. ^391,392 The lymphocytes from a typical cancer patient exhibit little or no cytolytic activity at baseline, but the cytolytic activity is increased by in vivo stimulation with IL-2, and the cytolytic activity is greatly enhanced by exposure to high concentrations of IL-2 in vitro. ^393,394 When PBMC are stimulated in vitro with IL-2, the resulting cell population is referred to as lymphokine-activated killer (LAK), and exhibits greater cytolytic properties than unstimulated NK cells. There is a subset of NK cells that are CD16- and bright CD56+ that express the high affinity IL-2 receptor and are exquisitely sensitive to IL-2. Activation of the more typical CD16+ and weak CD56+ require prolonged exposure to much higher concentrations of IL-2. The difference between NK and LAK cannot be defined phenotypically, but rather is defined by cytolytic activity using chromium-51 release by K562 and Daudi targets before and after stimulation by IL-2. NK activity is defined by cytotoxicity against the K562 cell line, but not the Daudi cell line, while LAK activity is defined by activity against both. Because LAK cells exhibit greater cytotoxicity against a broader range of malignant targets than unstimulated NK, clinical efforts have focused on LAK cells rather than NK cells.

Initial pilot studies in cancer patients seemed to confirm animal studies that demonstrated only limited antitumor activity from LAK cells alone in vivo. ³⁹⁵ Infusion of LAK cells was associated with fever, chills, and rigors because of the release of various cytokines from the activated cells. The continued presence of high concentrations of IL-2 was needed to retain the increased cytolytic activity, so LAK cells were infused concurrently with IL-2 in the early single-institution trials that were associated with a high objective RR. ²⁴² Clinical evaluation of LAK cells focused on combining LAK with IL-2, using either a high-dose bolus schedule, ^243,244 or a high-dose continuous-infusion schedule that delivered somewhat less IL-2 compared to the bolus schedule. ^245,396 Both approaches utilized 5 days of IL-2 to activate T-lymphocytes in vivo, then collection of lymphocytes by leukapheresis for additional stimulation with IL-2 in vitro, and then an additional 4–5 days of IL-2 in association with infusion of LAK cells. Hybrid approaches that utilized high-dose bolus IL-2 during the priming phase and continuous IL-2 after infusion of LAK did not appear to afford any additional benefit or less toxicity. ^{397 –399} A number of methodologic improvements took place that took advantage of cell separation technology and the use of plastic bags rather than roller bottles for expansion of LAK cells. ^{400 –402}

Very encouraging results, especially in patients with melanoma and renal cell cancer, were reported from single-institution studies conducted at the NCI, ²⁴⁴ at a community hospital, ²⁴⁵ and at a university hospital. ³⁹⁶ However, subsequent multicenter trials failed to confirm such high RRs, ^{249,403 –406} which suggested that unintentional patient-selection factors were responsible for the higher RR from the single institutions. Results in tumor types other than melanoma and renal cell cancer were also disappointing. ^{406 –409}

Of particular importance were three small randomized trials that were unable to show an advantage for IL-2 plus LAK compared to IL-2 alone. ^{262 –264} However, none of these trials were large enough to detect even a 50% difference in survival for a specific tumor type. The first trial randomized 23 renal cell and 20 melanoma patients to continuous infusion IL-2 with or without LAK cells. ²⁶² There were no responses in either arm in either patient group. In another trial, 71 renal cell cancer patients were randomized to a low-dose continuous infusion of IL-2 with or without LAK cells. ²⁶⁴ The overall RR was only 6% even though median survival was 13 months, and there was no difference in RR or survival. The largest study randomized 181 patients with various tumor types to a high-dose bolus IL-2 with or without LAK cells. ²⁶³ Differences were not significant, but the RR was slightly higher in the LAK group as a whole—24/85 versus 16/79 (p = 0.23)—and survival favored the LAK group (P₂  = 0.089). Subset analyses by tumor type lacked sufficient power to detect anything other than large differences in survival. In the 97 patients with renal cell cancer, there was no difference in response (33% with LAK, 24% without) or survival (2-year survival 47% with LAK versus 40% without, p = 0.52). However, for the 54 patients with melanoma, survival favored the patients who received LAK (2-year survival 32% versus 15%, p = 0.064) even though there was no difference in RR (22% with LAK versus 27% without). On the basis of these small randomized trials, and increasing evidence that IL-2 alone was an active agent, interest in LAK cells as a systemic therapy waned rapidly after approval of IL-2 alone for the treatment of renal cell cancer in 1992. Although many patients who received IL-2 and LAK had encouraging survival, it was impossible to determine what role, if any, LAK had in the outcomes. ^{246,247,250,254}

There has been some interest in the regional use of LAK cells, but few trials of any size have been reported. LAK cells have been given into the intraperitoneal cavity for ovarian cancer and other malignancies, ^410,411 into the hepatic artery for hepatocellular carcinoma ^412,413 and metastases from colorectal cancer, ⁴¹⁴ gastric cancer ⁴¹⁵ and melanoma, ^416,417 and into the pleural space for mesothelioma, ⁴¹⁸ and lung cancer. ^419–420 The largest interest for regional therapy has been generated for malignant gliomas and especially glioblastoma for which encouraging survival rates have been reported in trials with 19–40 patients. ^{421 –423} A randomized trial in progress is attempting to compare intraoperative placement of LAK to carmustine impregnated wafers in patients with glioblastoma.

The benefit of LAK cells as a monotherapy has never been systematically explored. Although the antitumor activity of LAK has been well-established, there have been no convincing clinical trial data to show that adding i.v. infusions of LAK cells enhances the benefits associated with systemic administration of IL-2. It is possible that larger randomized trials will confirm a survival benefit for the addition of LAK cells to IL-2 in the treatment of metastatic melanoma, but this would add substantial complexity and cost to the IL-2-based treatment. The opportunity for such a trial has probably passed. Increased understanding of immune signaling might lead to identification of other cytokines, ligands, agonists, or inhibitors that might be worth combining with NK cells in vitro and/or in vivo, or with LAK cells in vivo, because the activation of NK cells by non–HLA-restricted mechanisms remains an attractive treatment strategy.

Tumor-infiltrating lymphocytes and cytotoxic T-lymphocytes

For decades reports have suggested that patients whose tumors were heavily infiltrated with lymphocytes have better prognoses. ⁴²⁴ Some of the lymphocytes infiltrating tumors have specific antitumor activity. Tumor-infiltrating lymphocytes (TIL) are derived from primary or metastatic tumors, or regionally draining lymph nodes, and are enriched for T-lymphocytes and expanded in vitro in media containing IL-2. Chromium-release assays demonstrated that TIL could have as much as 10–100 times more cytolytic activity than autologous LAK cells after IL-2 stimulation. Some of the cells may represent cytotoxic T-lymphocytes (CTL), which are cells that react with tumor antigens in the context of HLA-1 presentation. Efforts were made to utilize TIL as a cancer therapy in combination with IL-2. ⁴²⁵ The original goal was to isolate and expand cytotoxic CD8+ cells that would be HLA class I restricted and only kill autologous tumor cells, ⁴²⁶ but most trials have utilized whatever final product emerged after culturing in IL-2 for several weeks.

The first report of TIL production described success in 24/25 patients, ⁴²⁷ but success rates of ∼70%–75% have been more representative as summarized in Table 4. ^{428 –442} Although most of the published experience has been with melanoma samples, the success rates are similar for a variety of different tumor types from both primary tumor tumors and metastastases. Early methods utilized roller bottles and 3-L semipermeable bags to expand cell numbers to >10¹¹, but, later, bioreactors were used to save space and expedite cell expansion. In practice, TIL stimulation results in cells that are predominantly CD8+, but sometimes are predominantly CD4+ or a more evenly mixed phenotype. The cells may be cytolytic or not, but when they are cytolytic, they are often HLA-restricted, in contrast to LAK cells.

Almost all trials of TIL have combined the cells with IL-2, and often with cyclophosphamide to decrease suppressor or T-regs. The first phase II trial describing the use of TIL with high-dose bolus IL-2 reported responses in 11/20 metatatic melanoma patients, ⁴³⁰ but the RR rate dropped to ∼33% after the treatment of 86 patients. ⁴³⁵ In contrast to results in animal models, RRs were similar regardless of whether or not cyclophosphamide was given just before treatment. ⁴³⁵ An RR of ∼25% was reported for pretreatment cyclophosphamide followed by TIL administered with high-dose continuous infusion IL-2. ⁴³³ Some patients had prolonged remissions and long term survival after receipt of IL-2 plus TIL. Other approaches with lower doses of IL-2 and no cyclophosphamide had RRs of 0%–20% in metastatic melanoma. ^{431,436,440,443} In metastatic renal cell cancer RRs ranged from 0% to 15% in most studies, ^{434,436,443,444} although 3 investigators reported RRs of 25%–35% from single institutions. ^431,445,446 None of these trials in renal cell cancer utilized high-dose bolus IL-2, and only one utilized high-dose continuous infusion IL-2 and cyclophosphamide. ⁴³⁴ Responses were observed in other tumor types, but large numbers of patients were never treated, and RRs were not encouraging outside of melanoma. Responders were more likely to have had more rapidly proliferating TIL cultures, to have received TIL that were cytolytic in vitro, and to have received larger numbers of TIL. Trafficking of radiolabeled TIL to depots of tumor was demonstrated for various tumor types in addition to melanoma, in both patients who had, and had not, received cyclophosphamide. ^{447 –449}

Even though most of the clinical work with TIL was in patients with melanoma, the only randomized, placebo-controlled, multicenter trial utilizing TIL in patients with metastatic disease was conducted in renal cell cancer. ⁴⁵⁰ The objective was to test whether IL-2+ TIL was superior to IL-2 plus placebo in patients with metastatic renal cell cancer who were candidates for nephrectomy, so that the primary tumor could be used to generate TIL. The trial utilized a low dose continuous infusion of IL-2 given daily 4 days per week for 4 weeks rather than higher doses of IL-2, and only 5 × 10⁹ to 3 × 10¹⁰ TIL. There were 178 patients enrolled, but only 160 were randomized because of histological and surgical issues, including a pathology diagnosis of other than renal cell cancer in 12 patients, insufficient tissue in 5, and unresectable growth in 1 patient. Of these 160 patients, 20 patients (9 randomized to receive TIL) were not medically fit to receive IL-2 following surgery; therefore only 140 patients actually received the first cycle of IL-2. The study was confounded further because the two central cell-processing laboratories were not able to produce TIL at the expected frequency. As a result, only 39 of the 81 patients were randomized to receive TIL (48%), and 59% of the 72 who started IL-2, actually received TIL. The trial was terminated early based on the recommendations of a data safety monitoring board when it became apparent that no significant differences would be detectable with the small study size. The study was analyzed only on an intent-to-treat basis with RRs of only 11% and 10%, and median survivals of 12.8 months and 11.5 months for the TIL and control groups, respectively. Although this was considered to be a negative trial, in reality, it was an inadequate trial, and the clinical efficacy of TIL has still never been tested. This trial illustrates the substantial logistical issues that confront randomized controlled trials that are attempted to test complex, patient-specific, cell products that take time to produce in a cell-biology laboratory.

An interesting randomized trial was conducted to test the benefit of giving adjuvant TIL with s.c. IL-2 in the postsurgical setting of resectable stage II and III non–small cell lung cancer (NSCLC). ⁴⁵¹ TIL were successfully established for 113/131 patients, and those 113 were then stratified by disease stage (II, IIIA, or IIIB) and randomized to TIL or standard treatment, which consisted of observation for stage II, and chemotherapy followed by radiation therapy for stage III patients. Median survival was better for the TIL patients (22.4 versus 14.1 months, p < 0.05). For the subsets, there was no difference for 39 stage II patients, but there was a benefit for TIL among the 42 patients with stage IIIA (p = 0.06) and the patients with stage IIIB disease (p < 0.01). This is a very interesting study that has not been confirmed or refuted.

Another randomized adjuvant trial was conducted in patients with stage III (node-positive) melanoma. ^452,453 There were 88 patients randomized to IL-2 plus TIL or IL-2 alone. After both 4 and 9 years of follow-up, there was no difference in PFS or OS. The subset of patients who had only a single positive lymph node did much better, compared to patients who had multiple positive lymph nodes. In the subgroup who had only a single positive node, at both 4 and 9 years of follow-up there was both a PFS (p = 0.02) and OS (p = 0.01) benefit associated with TIL. This observation would require confirmation in a large randomized trial.

Another randomized trial compared post surgical TIL plus IL-2 to chemotherapy in patients who had undergone resection of hepatic metastases from colorectal cancer. ⁴⁵⁴ Only 47 patients were randomized, and only 28 actually received the protocol-directed therapy (14 in each arm). There was no difference in PFS or OS.

Investigators at the NCI observed that the TIL from patients who had objective benefit were those whose TIL recognized certain antigens that were shared among different melanoma tumors. ^455,456 Subsequently, the researchers focused on subcultures of TIL that consisted of CTL restricted by tumor antigen and HLA-1 antigens, and then expanded those cells for therapy. Only one response was noted among 12 patients who had been immunized with gp100 prior to growing clones of CTL from tumors or peripheral blood, which were then infused with IL-2. ⁴⁵⁷ There were no responses among 9 patients treated with such vaccine-primed CTL following myeloablative therapy. ⁴⁵⁸ However, 18/35 patients with metastatic melanoma experienced dramatic and durable clinical responses following the same myeloablative therapy of cyclophosphamide and fludarabine to eliminate regulatory T-cells, followed by infusion of large numbers of tumor-reactive TIL. ^459,460 The response rate increased to 18/25 for the same treatment when 12 Gy of total body radiation was added to the myeloablative chemotherapy regimen. ⁴⁶¹

A number of laboratories have utilized anti-CD3 antibodies in combination with IL-2 in the process of generating TIL because it expedites expansion. ^{432 –434,436,446,462} Recently there was a single-institution report of a 50% RR in 20 patients with metastatic melanoma who were treated with short-term TIL cultures utilizing this approach in combination with nonmyeloablative therapy and high-dose bolus IL-2. ⁴⁶³ It may also be important that these patients had received GM-CSF to mobilize hematopoietic stem cells prior to undergoing the myeloablative therapy. If verified, this approach might be somewhat a less-expensive and more-rapid approach than that used at the NCI.

TIL continue to be of great interest, although it is now appreciated that the cellular microenvironment also may promote tumor survival and/or inhibit immune cells that recognize the tumor. ^464,465 Such immune-cell interactions are not confined to the tumor itself which may explain the benefits of giving cytotoxic therapy to reduce or briefly eliminate regulatory T-cells and suppressor cells that inhibit an immune cellular response.

CD3-driven helper T-lymphocytes

This adoptive cell therapy approach has utilized activation via CD3, rather than IL-2 activation via CD25, to stimulate immune cells. ^{466 –470} This approach was originally referred to as autolymphocyte therapy (ALT), ⁴⁶⁷ and such cells also were termed autologous activated lymphocytes (AAL), ⁴⁷⁰ but neither of these terms is a useful descriptor. The cells included in this category are CD4+ CD25− CD45RO+ memory helper T-cells. CD4+ helper T-cells may stimulate cytotoxic T-cells and antitumor antibody production by B-cells. Memory helper T-cells are a common component of tumor infiltration of primary renal cell carcinoma. ⁴⁷¹ Their presence in locally invasive colorectal cancer is associated with a better survival compared to patients whose tumors lack such cells. ⁴⁷² Such cells can induce autoimmunity against tumor-associated antigens, ⁴⁷³ but are inhibited by CD4+ CD25+ T-regulatory cells. ⁴⁷⁴

Because of evidence that CD8+ suppressor and regulatory T-cells can inhibit an immune response, adoptive cell therapy with this approach has usually included other immune-modulating agents in the treatment strategy. Most of the trials with anti-CD3 activated helper T-cells have utilized cimetidine as a putative inhibitor of CD8 suppressor T-cells, and possibly CD4+ regulatory T-cells rather than more toxic approaches such as chemotherapy. ⁴⁷⁵ There is evidence from small randomized trials that preoperative cimetidine is associated with increased numbers of tumor-infiltrating lymphocytes at the time of surgical resection in certain tumors, ^{476 –478} but apparently not others. ^479,480

The first step for production of these memory helper T-cells was collection of PBMC by leukoapheresis, followed by in vitro procedures to enrich for CD4+ CD25- helper T-cells. An initial leukapheresis was used to collect autologous lymphocytes, which were stimulated with anti-CD3 mAbs to secrete cytokines. The resulting cytokine-enriched media contained significant amounts of TNF-α, IL-1β, interferon-γ, and IL-6, but not IL-2. Residual murine anti-CD3 mAb was also detectable in this media. Incubation in this media of subsequently collected PBMC resulted in a cell population that consisted primarily of CD4+, CD25–, CD45RO+ cells, characterized as memory helper T-cells rather than the phenotype of NK, NKT, or LAK cells. In contrast to LAK cells and NK cells, these cells are not cytotoxic. Patients receiving infusions of these cells also received very high doses of cimetidine (600 mg four times a day) to inhibit suppressor/regulatory T-cells. Treatment protocols utilized only 10⁹ cells per treatment, which were well-tolerated in the outpatient setting.

In clinical trials, an encouraging median survival of 15 months and a 2-year survival rate of 36% was reported for 36 patients with metastatic renal cell cancer. ⁴⁶⁶ What was even more impressive was a multicenter 90-patient randomized trial in renal cell cancer in which cimetidine plus ALT was superior to cimetidine alone in terms of RR (21% versus 5%), and median OS (20 versus 8 months, p = 0.008). ⁴⁶⁷ However, in a 335-patient community-based phase II trial, in which ALT was provided by one of three different treatment centers, an RR of only 4% was documented for 259 evaluable patients, and survival ended up being disappointing. ⁴⁶⁸ In addition, a small multicenter phase II trial performed by the Cancer Biotherapy Research Group using the same procedure failed to confirm a promising objective RR in renal cell or colon cancer. ⁴⁷⁰ The company Cellcor subsequently sponsored a 180 patient multicenter randomized trial of ALT with cimetidine versus INF-α in patients with metastatic renal cell cancer in which the researchers were unable to show an advantage for the cell therapy. Results of that trial were never published and the company was acquired by Cytogen and Neoprobe and ceased operations.

There has been some renewed interest in approaches with CD4-positive cell populations based on more recent insights into the complexity of tumor immunology. A similar product called Xcelerated T-cells (Xcyte Therapies, Seattle, WA) was produced by incubating autologous peripheral blood cells from which monocytes were removed with anti-CD3 and anti-CD28 antibodies for 8 days, then reinfusing the cells followed by 10 days of s.c. IL-2. ⁴⁸¹ Approximately 20 billion cells were infused each time, and some patients were treated more than once. The final cell products were 64% CD3+, CD4+, and 25% CD3+ CD8+. There were 26 patients treated, and almost all received two courses of treatment. Although no ORs were reported, the median survival of 21 months suggests either a very favorable patient population or a benefit that was not associated with objective regression in tumor size. Results from subsequent studies in myeloma and CLL have not been encouraging enough to publish.

At this time, there is no accepted role for adoptive cell therapy with memory/helper T-cells. However, it is noteworthy that at least two companies have pursued potential commercialization of such noncytotoxic T-lymphocytes because, theoretically, IL-2 is not needed for therapeutic benefit. The trials that have been performed suggest there may be some activity in renal cell cancer, but other modifications of the therapeutic approach and larger randomized trials would be needed to establish such a therapy.

Nonmyeloablative allogeneic stem-cell transplant and donor lymphocyte infusions

For more than 3 decades, we have known that part of the therapeutic benefit from allogeneic transplants the result of graft-versus-host disease (GVHD), which includes a graft-versus-leukemia effect in leukemia patients. ^482,483 This was established by showing that patients who received haploidentical hematopoietic stem cell transplants from identical twins had less GVHD than patients who received allogeneic hematopoietic stem-cell transplants, but the latter had a longer progression-free interval. While severe GVHD can be life-threatening or lethal, mild-to-moderate GVHD is associated with a better survival than no GVHD. ⁴⁸⁴ GVHD and graft-versus-leukemia are both mediated by donor T-lymphocytes. ^{485 –487}

Historically myeloablative allogeneic transplants have been associated with high morbidity and mortality rates because of combinations of failure of the allogeneic transplanted cells to engraft, end organ toxicity caused by chemotherapy and/or radiation therapy, and severe GVHD. For this reason, increasing use is being made of nonmyeloablative transplants (so-called “mini-allos”) in which immunosuppressive cytotoxic chemotherapy is used to suppress the host immune system to permit engraftment of an HLA-matched allogeneic immune system for the purpose of producing a graft-versus-tumor effect, rather than total elimination of all host hematopoietic cells. ⁴⁸⁸ This state of mixed chimerism with persistence of both donor and host hematopoietic and lymphoid cells limits GVHD, but retains substantial graft-versus-tumor effects. ^{489 –492} If complete donor chimerism is not achieved after such a nonmarrow ablative transplant, then donor lymphocyte infusions (DLIs) can be administered along with immunosuppressive therapy agents such as methotrexate, antithymocyte globulin, cyclosporine, tacrolimus, and mycophenolate mofetil, to create an environment in which lymphocytes from the same HLA-matched marrow donor can survive. ^492,493 In such a chimeric immune system, DLI can still be cytotoxic to tumor cells with minor differences in histocompatibility alloantigens or against tumor-associated antigens. In the absence of such chimerism, infusions of allogeneic peripheral blood lymphocytes are typically eliminated by the host immune system.

Nonmyeloablative transplants have been used increasingly in hematologic maligiancies, especially in elderly patients. ^488,494 The use of nonmyeloablative allogeneic transplants and selective use of DLIs has been accepted as standard therapy in a variety of hematologic malignancies. Most of the experience has been in CML ^{495 –499} and the acute leukemias. ^{500 –507} This approach is associated with a high rate of complete molecular remissions and is now considered appropriate for CML patients for whom two or more tyrosine kinase inhibitors have failed. ⁴⁹⁹ In AML the approach was associated with a 5-year survival rate of 33% for 274 patients with a median age of 60. ⁵⁰⁷ Results have not been very good in patients with relapsed ALL. ^500,503 There have also been reports of its applications in most other hematologic malignancies including B-cell lymphoma, ^508,509 T-cell lymphoma, ^510,511 Hodgkin's disease, ⁵¹² multiple myeloma, ^{513 –515} and CLL. ^516,517 Long-term follow-up of 102 patients with myeloma revealed a 5-year OS of 65% and a 36% PFS. ⁵¹⁵ Long-term follow up of 82 patients with CLL revealed a 5-year survival rates of 50% and a PFS of 39%. ⁵¹⁷ In these trials, ∼25% of patients died from failure to engraft, GVHD, and/or opportunistic infections, but GVHD was associated with better survival, which is consistent with the presence of a graft-versus-malignancy effect.

Nonmyeloablative transplants and DLI also have been tested in solid tumors, but the results have not been as encouraging as in the hematologic malignancies. ^{518 –521} Most of the experience has been in patients with metastatic colorectal, ⁵²¹ breast, ^{522 –536} and renal cell cancers. ^{527 –537} Smaller numbers of patients with other malignancies have also been treated. ^{521,537 –539} Most centers have been able to treat only a small number of patients. Most deaths have been from progressive cancer, but early deaths resulting from treatment-related complications were also common. In colorectal cancer, a multicenter trial of 39 patients was associated with a 20% RR and a 10% treatment-related mortality. ⁵²² The largest experience in breast cancer is a multicenter 66-patient trial in which 39 patients had myeloablative and 27 nonmyeloablative therapy, but the latter group had worse performance status and a lower rate of GVHD, and a 1-year PFS of only 8%. ⁵²⁵

The first report that attracted widespread attention was based on 10/19 patients with metastatic renal cell cancer having an objective response to nonmyeloablative allogeneic transplantation at a single institution. ⁵²⁷ Thereafter ensued a flurry of studies investigating this approach in that patient population (Table 5). ^{527 –537} There were no objective responses in a 22-patient multicenter U.S. cooperative group trial. ⁵³⁵ The largest experience so far published is a European multicenter 124-patient trial in which 42 patients received DLI. ⁵³⁶ The RR was 24/124 (19%) and the 1-year transplant mortality rate was 16%. Median follow-up was only 15 months, but patients with chronic GVHD who also received DLI had a projected 2-year survival of 70%.

The benefits of this approach appear much less impressive in solid tumors than in the hematopoietic malignancies. Acute and chronic GVHD remain problematic in all settings, but are also associated with response and long-term benefit. Performance status, tumor burden, and chemotherapy resistance are all important variables, especially in patients with solid tumors. As typically happens, RRs have diminished as more patients have been treated, and the proportion of patients that have had CR is disappointing. However, proof-of-principle has been clearly established, and some patients have obtained remissions that have lasted for many years. Most patients have had standard therapies that failed before accepting the risks associated with this approach. There is still a potential role for other immune-modulating agents or cell-selection procedures to improve the original approaches.

Sipuleucel-T

In 2010, sipuleucel-T (Provenge®) became the first cell-based therapy other than hematopoetic stem cell transplantation, to be sanctioned for the treatment of cancer, specifically for hormone-refractory prostate cancer. This product was conceptually developed to be vaccine or an active specific immunotherapy. This cell product (APC8015) is produced by collecting autologous PBMC that are incubated for 3–3.5 days with a recombinant fusion protein PA2024, that consists of prostatic acid phosphatase (PAP) fused to GM-CSF. ^540,541 Release criteria for the therapeutic product include the presence of at least 40 million cells that express the activation molecule CD54 (ICAM). The original concept was that, in the presence of the GM-CSF portion of the fusion molecule, monocytes and macrophages would become dendritic cells and become loaded with PAP as an antitumor antigen. However, the final cell product includes a variety of leukocytes including B-lymphocytes, T-lymphocytes, monocytes, and macrophages rather than just antigen-presenting cells, and also includes the fusion protein. The cell mixture is infused i.v. ∼3–4 days after the pheresis procedure. In the pivotal trial the treatment was given every 2 weeks for three total treatments over ∼1 month. ⁵⁴² Because of the mixture of cells, the precise antitumor mechanism is not clear, although there is evidence that many patients do mount both a humoral and cellular immune response to the PA2024 fusion protein. Because it is a heterogenous cell product that is being administered i.v., for this review, it is considered an adoptive cell therapy rather than a vaccine.

Infusions of sipuleucel-T have been well-tolerated, with the most frequent adverse events being mild flulike symptoms, including chills, fever, and asthenia. ^{542 –546} Occasionally, dyspnea, nausea, and vomiting have occurred within hours after completion of the cell infusion. These infusion-related reactions are probably the result of cytokine release by activated cells. In the large randomized pivotal trial that led to approval of sipuleucel-T, the rate of chills was 54% in the activated-cell arm versus 12% in the control arm, which included a smaller number of unactivated cells. ⁵⁴²

In pilot studies 100% of patients with hormone-refractory prostate cancer, who received APC8015, had an immune response to the fusion protein, and 38% developed an immune response against PAP. ⁵⁴³ Three (3) patients had a >50% decline in prostate specific antigen (PSA) tumor marker. A phase II trial was carried in 19 patients out, utilizing two infusions followed by three monthly s.c. injections of 1.0 mg of PA2024. ⁵⁴⁴ One (1) patient had a durable CR that lasted more than 4 years. This response included normalization of a pretreatment serum PSA level of 221 ng/mL at 4 months after starting treatment and eventual resolution of metastatic retroperitoneal and pelvic adenopathy. Two (2) other patients had transient 25%–50% decreases in PSA.

A placebo-controlled randomized trial (D9901) that utilized three infusions every 2 weeks was conducted with PFS as the primary endpoint. ⁵⁴⁵ A total of 127 patients with hormone-refractory prostate cancer were randomly assigned in a 2:1 ratio to receive sipuleucel-T (n = 82) or placebo (n = 45). To facilitate accrual, at the time of disease progression, patients who had received the placebo, which consisted of autologous PBMC that had not been incubated with PA2024, were eligible to receive a single i.v. infusion of the remainder of their cryopreserved cells after the cells had been incubated with PA2024 to create APC8015F (F for frozen). Analysis was performed after all patients had been at risk for 3 years, by which time only 12 patients had not died or experienced progressive disease. The median TTP was 11.7 months for sipuleucel-T compared with 10.0 months for the placebo arm (p = 0.052). The median overall survival was 25.9 months for the active treatment compared with 21.4 months for placebo. (p = 0.01) T-cell stimulation at 2 months was eightfold higher in the patients treated with sipuleucel-T.

A second randomized trial with the same design (D9902A) failed to find a difference in either TTP or OS in 98 patients. The data from this trial were combined with those from the first phase III trial resulting in an analysis of 147 patients who received sipuleucel-T and 78 who received placebo. ⁵⁴⁶ There was a 33% reduction in the risk of death and median survival was longer for the cohort treated with sipuleucel-T (p = 0.011), but there was still no difference in TTP. There was a correlation between CD54 upregulation and overall survival. The FDA declined to approve sipuleucel-T based on this data, because the primary endpoint, PFS, had not been met.

Meanwhile, a third randomized trial completed enrollment with OS as the primary endpoint rather than TTP. ⁵⁴² This multicenter, double-blind, placebo-controlled trial accrued 512 patients with a median age of 71 between August 2003 and November 2007. The patients were randomized using the same 2:1 ratio resulting in 341 patients receiving the sipuleucel-T and 171 receiving placebo. Each sipuleucel-T product had at least 40 million cells that expressed CD54; they were infused i.v. over 60 minutes. The placebo product consisted of one third of the collected cells incubated in medium without the PA2024. Ninety-two percent (92%) of patients got all three planned infusions, which were administered every 2 weeks for three doses. At the time of disease progression, patients who had received placebo had the option to receive the APC8015F product derived from their residual cryopreserved PBMC and incubated with PA2024. For 84 patients (49%), the thawed product was the first treatment received at relapse; eventually, 109 (64%) relapsing patients received APC8015F. Patients randomized to sipuleucel-T had a longer OS (25.8 versus 21.7 months; p = 0.02) and a 22% decrease in the risk of death (p = 0.03). Subsequent treatment with docetaxel did not explain the difference in survival. Antibody titers against PA2024 exceeded 400 in 100/151 (66%) of the sipuleucel-T cohort, compared to 2/70 (3%) for placebo. Elevated antibody titers against PAP were documented for 43/151 (28%) for the active product versus 1/70 (1%) for the placebo. At week 6, T-cell proliferation was increased in 46/63 (73%) of sipuleucel-T patients versus 4/33 (12%) for control patients and T-cell responses to PAP were noted in 15/55 (27%) versus 2/25 (8%). The humoral response correlated with survival, but the cellular response did not.

It is anticipated that men with hormone-refractory metastatic prostate cancer will welcome this product as long as payers cover most of the charges associated with the treatment. Medical oncologists may still prefer docetaxel as the first systemic therapy for such patients for a variety of reasons of convenience and practice revenue. A head-to-head comparison of this immunotherapy versus docetaxel chemotherapy would be interesting but probably will not be conducted.

Vaccines for melanoma

Melanoma has been the primary target of numerous cancer vaccine efforts. Table 6 summarizes the results of a number of randomized trials that have been conducted in melanoma. By and large, the results have been disappointing. The antigenic sources included in these vaccines were disialogangliosides, ^151,549 allogeneic tumor cell lysates, ^{171,550 –553} allogeneic tumor cells, ⁵¹ heat-shock proteins derived from autologous tumor, ⁵⁵⁴ and HLA-restricted peptides. ^370,554 It was particularly disconcerting that, in many trials, vaccines have been inferior to other biologicals such as IFN-α, ¹⁵¹ BCG alone, ³⁷⁰ or the anti-CTLA-4 mAb ipilimumab. ⁵⁵⁵ In recent years, many trials have focused on peptide antigens and were restricted to HLA-2 positive patients. One trial appeared to have a positive result at the time of abstract presentation, but at the time of this review, had not yet been published. During the years 2000–2007, 185 HLA-2 positive patients with metastatic melanoma, who were healthy enough to receive high-dose bolus IL-2, were randomized to receive either high-dose bolus IL-2 alone or in combination with the gp-100 peptide vaccine. ³⁷⁰ There appeared to be a benefit from the addition of gp-100 in terms of PFS but not OS. The results of a large, randomized, placebo controlled 4-arm trial that compares the gp100 vaccine to placebo, with and without GM-CSF, in HLA-2 positive melanoma patients with completely resected stage III or IV disease, has not yet been reported.

A number of smaller exploratory randomized trials have examined other aspects of melanoma vaccines. One group explored different doses of BEC2, an anti-idiotypemAb that reacts with the hypervariable regions of an anti-GD3 gangliosidemAb. ⁵⁵⁶ This group has also administered this BEC2 and the GD3 gangliosides in a sequential therapeutic strategy. ⁵⁵⁷ Groups working with various peptide vaccines have confirmed that immune responses against the peptides can be induced as measured in elispot and tetramer assays, but these were associated with minimal if any antitumor effects. ^{558 –566} The focus on monopeptide vaccines has been useful for proof-of-principle in trials that have confirmed that vaccination with such products can enhance antigen-specific immune responses, but not all melanoma patients have tumors that express the specific peptides, and it is unclear whether any single peptide target is sufficient for a significant antitumor effect. Limiting trials to patients whose tumors express large quantities of such peptides may be needed to get positive clinical effects. Polyvalent or cell-based vaccines may be more clinically relevant. Vaccines containing multiple peptides are probably superior to fewer peptides or monopeptide products. ⁵⁶²

A number of small randomized trials have explored the potential benefit of different adjuvants. In one trial there was no difference whether GM-CSF or IFN-γ was administered at that same time as injections of irradiated tumor cells derived from proliferating autologous tumor-cell lines. ⁵⁶⁷ Small randomized studies have failed to show a benefit for adding various putative immune-enhancing adjuvants such as monaside, ⁵⁶⁴ IFN-α, ⁵⁶⁶ IL-2, ⁵⁶⁸ or GM-CSF. ^{558,563 –566}

Theoretically, vaccines directed against proliferating progenitor or stem cells may be superior to these other approaches for a variety of reasons. ⁵⁶⁹ A 5-year survival rate of more than 25% was reported for 74 patients treated with a vaccine consisting of irradiated proliferating autologous tumor cells. ⁵⁷⁰ Long-term PFS and a 5-year OS rate of greater than 50% was observed in patients treated with patient-specific vaccines consisting of autologous dendritic cells loaded with such proliferating tumor cells. ^571,572 A randomized trial is in progress that compares these two different products in metastatic melanoma patients.

Idiotype vaccines for B-cell malignancies

The most tumor-specific tumor antigen known is the idiotype of the paraprotien of a monoclonal B-cell lymphoproliferative disorder. For this reason, there has been a long-standing interest in the idiotype as a target for patient-specific treatment of B-cell lymphoma. In fact, patient-specific murine anti-idiotype mAbs produced long-lasting antitumor responses in a number of patients. ^{573 –577} The goal of immunization with idiotype is production of endogenous human anti-idiotype antibodies as well as idiotype-specific CTL responses. Hybridoma and recombinant DNA techniques have been used to rescue the idiotype from fresh lymphoma cells. The idiotype is then mass-produced for therapeutic use as a patient-specific therapy. Most of the efforts have focused on patients with indolent lymphomas, particularly follicular lymphoma, because of the higher probability of success in rescuing the idiotype, ⁵⁷⁸ because of the slower proliferative rate of such tumors, ⁵⁷⁹ which allows time to rescue the idiotype and prepare the vaccines, evidence that some follicular lymphomas harbor tumor reactive T-cells, ⁵⁸⁰ and currently there is no curative therapy for this entity.

Pilot studies involving nine patients established that endogenous humoral and cellular anti-idiotype responses could be induced by vaccinating with idiotype, and 2 patients with measurable disease experienced a CR. ⁵⁸¹ Eleven (11) of the first 16 patients had a T-cell response after vaccination, and those patients appeared to have a longer PFS. ⁵⁸² Of 41 patients with indolent B-cell lymphoma, 49% had anti-idiotype immune responses following vaccination with idiotype conjugated to keyhole limpet hemocyanin (KLH). ⁵⁸³ In the subset of 32 patients who were in remission after their first treatment, TTP was longer in patients who exhibited an anti-idiotype immune response, compared to those who did not (7.9 versus 1.3 years, p = 0.0001). Complete molecular remissions were documented in some patients, ⁵⁸⁴ and long-term follow-up confirmed that such responses could be quite durable. ⁵⁸⁵ An idiotype vaccine was prepared for 83% of 33 consecutive patients with follicular lymphoma in whom a clinical remission had been induced with doxorubicin-based chemotherapy after first relapse following an original complete response to chemotherapy. ⁵⁸⁶ Twenty-five (25) patients were treated, with 20 having an immune response that included a humoral idiotype-specific response in 13 patients, and a cellular idiotype-specific response in 18 patients. Each of the 20 immune-responders had a longer DFS after a second remission than the first remission.

An analysis of 136 patients treated with patient-specific idiotype vaccines in various trials carried out at Stanford showed that patients who had a humoral response had a longer PFS, compared to those who did not (8.2 versus 3.4 years, p = 0.018) and that Fc polymorphisms were an independent predictor of outcome. ⁵⁸⁷ Patients with the FcgammaRIIIa 158 valine-valine (V/V) genotype had a longer disease-free interval after idiotype vaccine treatment, compared to those with valine-phenylalanine (V/P) or phenylaline-phenylalanine (P/P) genotypes. A multicenter 21-patient study confirmed that 62% of patients developed an anti-idiotype immune response after treatment with idiotype linked to KLH, and injected s.c. with GM-CSF. ⁵⁸⁸ Other variations of idiotype immunization approaches have included suspending the idiotypes in liposomes with IL-2, ⁵⁸⁹ pulsing autologous dendritic cells with idiotype, ⁵⁹⁰ and use of plasmid idiotype DNA. ⁵⁹¹

The encouraging results with idiotype vaccination attracted commercial interest and one positive randomized trial has been reported. As of late 2010, there were no idiotype vaccines that had received regulatory approval, but three products, MyVaxID, ⁵⁹² FavID, ⁵⁹³ and BiovaxID ⁵⁹⁴ had been tested in phase III pivotal trials in patients with follicular lymphoma. As summarized in Table 7, all three products utilized the strategy of linking idiotype to KLH and administering the vaccine with GM-CSF following remission to standard therapy. Biovax was produced by hybridoma rather than recombinant DNA technology. What is perhaps most important is the significant differences in the patient populations enrolled in these phase III trials—most notably, the requirement for a CR in the BiovaxID trial while patients having either a PR or CR were eligible for the MyVaxID and FavID products. In 2008, efforts to gain regulatory approval for the first two products, MyVaxID and FavID, were discontinued when pivotal trials failed to meet any of their PFS endpoints.

In contrast to these two negative trials, the positive results of a phase III trial for BiovaxID were presented at the plenary session of the 2009 annual meeting of the American Society for Clinical Oncology. ⁵⁹⁵ Of 177 patients treated with PACE (prednisone, doxorubicin, cyclphophosphamide, and etoposide), 117 were still in complete remission and eligible for protocol-directed therapy in a 2:1 randomization. The 76 patients who received the idiotype vaccine had a longer TTP than 41 who received a placebo vaccine (44 versus 31 months, p = 0.045), but there was no difference in OS. Among the patients who received the idiotype vaccine, 95% had detectable anti-idiotype specific T-cell responses while 75% had anti-idiotype immunoglobulin responses.

Many researchers remain skeptical about this approach given the small study size and nonrobust p-value of the one positive trial, especially in light of the two negative trials with similar products, for which results may never be formally published. The failure to make an impact on survival is not surprising but suggests that this may also not be a curative therapy. The relevance is especially unclear, because, when these trials were designed, none used biochemotherapy induction. Rituximab combined with chemotherapy is the current standard induction treatment for follicular lymphoma and has been associated with higher CR rates and longer PFS when rituximab consolidation or maintenance is used.

Dendritic cell vaccines

Dendritic cells (DCs) are a discrete leukocyte population in the monocyte/macrophage family, that display antigen in the context of histocompatibility antigens to T-lymphocytes. ^596,597 DCs are believed to be the most important class of antigen-presenting cells (APCs) and are sometimes referred to as professional APCs. DCs are able to take in, process, and present antigen; are able to migrate through tissues; and are able to stimulate antigen-specific T-cell responses. DCs drive both CD4+ and CD8+ antigenic responses. Immature DCs are phagocytic and take up and process antigen, but as they mature their phagocytic capacity diminishes in association with antigen presentation. For this reason, most of the interest in DCs has been to present antigen for immunization or vaccination purposes for a primary response or to boost weak existing responses. Various cytokines optimize the production of DCs, especially the combination of IL-4 and GM-CSF. ⁵⁹⁸ Original isolation and enrichment procedures focused on cells that were adherent to plastic, but now elutriation procedures and equipment are available that have made it easier to standardize manufacturing of DC products.

DCs express a variety of cluster designation (CD) markers. CD11c, CD80 (B7.1), CD86 (B7.2), and CD83 are commonly used to define and characterize DCs and to differentiate between immature and mature DCs. B7.1 and B7.2 bind to CD28 for a costumulatory signal and to CTLA-4 for an inhibitory signal. Therefore, antigen-loaded DCs are capable of producing either an antiantigen immune response or tolerance. DCs can be derived from a variety of tissues, but most DCs being tested clinically are derived from PBMC obtained by leukapheresis without any in vivo stimulation by cytokines or other agents. In fact, pretreatment with GM-CSF may induce premature maturation and diminish antigen-loading capability. Animal experiments have suggested that i.v. infusions of DC are more likely to produce CD4+ immune responses, while s.c. injections are more likely to produce a CD8+ immune responses.

DCs have been administered by various routes. From a product-classification standpoint, pure DC populations that have not been antigen-loaded, would be considered as an adoptive immunotherapy. DCs that are antigen-loaded and administered by s.c., i.d, intranodal, or lymphatic routes of administration, are considered to be vaccines. Such products infused i.v. could be classified as either adoptive cell therapy or vaccine, but most investigators would consider such products vaccines if the DC population was pure. Heterogenous leukocyte preparations that are given i.v., even if there has been some antigen-loading process, are probably most appropriately classified as adoptive cell therapies, which is why, for this review, the sipuleucel-T product was included as an adoptive cell therapy, rather than a vaccine. There are no pure dendritic cell products that are of established benefit at this time, but there are numerous DC-vaccine products under investigation.

Ofatumumab

Ofatumumab (Arzerra®) is a human IgG1 κ anti-CD20 mAb that was developed because of the great success of rituximab in the treatment of B-cell malignancies. There is reason to believe that may be even more effective that rituximab because of stronger reactivity with human complement and effector cells to enhance CDC and ADCC, and a different binding site on the CD20 molecule. ^{730 –733} Ofatumumab was granted regulatory approval in October 2009 for the treatment of patients with CLL that was refractory to fludarabine and alemtuzumab. ⁷³⁰ Subsequently, rituximab has also been granted a marketing indication in CLL where it has been used with chemotherapy for several years. Even though CD20 is expressed on mature B-cells, it has proven to be an excellent target for anti–B-cell therapy, because it is expressed on most malignant B-cells, but not stem or plasma cells. It is often expressed at high levels on malignant B lymphocytes. Anti-CD20 mAbs are effective for a variety of reasons. ⁷³⁴ Because it is not internalized or shed from the plasma membrane following binding, mAb persists on the cell surface for extended periods of time allowing fixation of complement proteins that mediate CDC, binding to Fc by effector-cell binding via FcR. mAb binding to CD20 can also generate transmembrane signals that induce apoptosis, which is another mechanism for antitumor effects. ⁷³⁵ However, other researchers have suggested that type-1 mAbs, such as rituximab and ofatumumab, as opposed to type-2 mAbs, such as tositumomab, may depend more heavily on FcR mediated immunological effects. ⁷³⁶

Ofatumumab differs from rituximab in that it binds to an extracellular loop of CD20 that is opposite from the extracellular binding of rituximab, and ofatumumab also binds to the intracellular loop that passes through the cell membrane near the N-terminus of the protein. ^737,738 Compared to rituximab, ofatumaumab attaches closer to the cell membrane and binds CD20 with greater avidity, which increases exposure of the Fc portion of the mAb for binding to complement and/or effector cells to affect CMC and ADCC. ^{739 –741} The in vitro superiority of ofatumumab over rituximab is based on CDC assays. ^{737,738,740,742–743} Ofatumumab affects CDC more efficiently than rituximab against sensitive targets and produces activity against cells that express low levels of CD20 that are rituximab-resistant.

At this time, the results of only a few clinical trials with ofatumumab have been published, but one would expect ofatumumab to be active against the same B-cell malignancies as rituximab. In a dose-finding phase I/II trial in 40 patients with recurrent follicular lymphoma, a maximum tolerated dose was not identified over the range of 300– 1000 mg in four cohorts of patients who received four weekly infusions of ofatumumab. ⁷⁴⁴ In a dose-finding phase I/II trial in 33 patients with CLL, a maximum tolerated dose was not identified over the range of 100–2000 mg, using schedules that included a lower initial dose in an effort to minimize infusion reactions, followed by three weekly infusions at higher doses. ⁷⁴⁵ In the third dosing cohort, PRs were observed in 13/26 patients. Infusion reactions, especially with the first dose before circulating B-cells had been cleared, were the most common adverse events. A trial was conducted in CLL patients whose conditions were either considered refractory to fludarabine (n = 59) or consisted of bulky (>5 cm) lymphadenopathy (n = 79). ⁷⁴⁶ In this trial, 300 mg of ofatumumab was given initially, then 2000 mg weekly for 7 weeks, and then monthly for 7 months. RRs were 57% in the fludarabine-refractory cohort and 47% in the bulky-CLL group. These appeared to be much higher RRs then those reported for rituximab in even the front-line setting of CLL. The most common side-effects were grade 1 and 2 infusion reactions, which were especially common with the first treatment, even though patients were given 100 mg of prednisolone or equivalent prior to infusions 1, 2, and 9.

A head-to-head comparison of ofatumumab and rituximab would be interesting because of the apparent advantages of ofatumumab in terms of immunotherapy mechanisms. However, it is doubtful such a study will actually take place, in part, because it is unlikely that there would be dramatic difference in outcomes, especially when the most common use of rituximab is in combination with chemotherapy for which RRs and survival rates are so good, that it would be difficult to show additional benefit. Another issue is the definition of true rituximab resistance, because, for clinical trial purposes, patients who have a response to rituximab lasting less than 6 months are grouped with patients who actually progress while on therapy, and these patients may have very different mechanisms of resistance to CD20-based therapy.

Alemtuzumab

Alemtuzumab (Campath®) is a humanized anti-CD52 mAb that was originally approved in 2001 for the treatment of fludarabine-refractory CLL, but also has a marketing indication as monotherapy in the first-line treatment of CLL. ⁷⁴⁷ Alemtuzumab was the second mAb approved for the treatment of a hematologic malignancy. The original rat antibody Campath-1G was genetically altered to produce Campath1-H by joining the hypervariable region of the rodent antibody to the variable framework and constant regions of a human IgG1 κ. ^748,749 This construct became alemtuzumab. The human IgG1 Fc receptor resulted in enhanced CMC and ADCC in vitro, compared to earlier rat and murine mAbs to the same epitope and was more active in vivo. ^{750 –752} CD52 is not internalized after antibody binding, and it does not appear to be secreted. ADCC has been repeatedly shown to be at least one way for alemtuzumab to produce antitumor effects. ^749,753,754

In a study of 36 CLL patients who were treated with the standard i.v. dose and schedule, there was no correlation between response to alemtuzumab and and the genotype for the high-affinity, FcγR-receptor polymorphisms FcγR3A and FcγR2A. ⁷⁵⁵ There is also evidence that alemtuzumab can produce antitumor effects via alteration of intracellular signal transduction. ⁷⁵⁶ CD52 is not only expressed by most lymphomas and chronic lymphoid leukemias but also by normal B- and T-lymphocytes, NK cells, monocytes, macrophages, dendritic cells, neutrophils, and mast cells. ⁷⁵² Human neutrophil binding to alemtuzumab is associated with complement lysis of the neutrophils, which contributes to infusion-related toxicity and the neutropenia that occurs in some patients. ⁷⁵⁷

Because it binds to circulating B- and T-lymphocytes and monocytes, i.v. infusions of alemtuzumab are associated with significant infusion reactions. ^752,758 Because of this, low doses are given initially at slow rates. Once circulating CD52 cells have been cleared, higher doses and faster rates of infusion can be utilized. The s.c. route minimizes the initial reaction and enables a more rapid achievement of sustained serum levels with similar antitumor effects. ^759,760 Almost all patients treated by the s.c. route experience significant local skin reactions that can be severe. Because of this agent's profound effects on both B- and T-lymphocytes, treatment with alemtuzumab is typically associated with a long duration of immunosuppression and risk of opportunistic infections. ^751,761 Prophylactic antivirals are effective for reducing the risk of activating cytomegalovirus infection. ⁷⁶²

Alemtuzumab was originally approved in 2001 based on a 33% RR in patients with fludarabine-refractory CLL. ⁷⁵⁸ In the CLL trials, the dose of alemtuzumab was gradually increased from 3 mg to 10 mg, and then to 30 mg, thrice weekly. In the relapsed setting, numerous other reports have described RRs ranging from 30% to 50% and durable remissions averaging 6–12 months in duration when administered by either the i.v. ^{763 –765} or s.c. routes. ^{766 –769} RRs in the range of 80%–90% have been reported in previously untreated CLL patients. An RR of 87% was achieved by s.c. administration of alemtuzumab and prolongation of treatment from 12 to 18 months. ⁷⁶⁹ In a randomized trial of initital therapy in 297 CLL patients, i.v. alemtuzumab produced a higher RR than oral chlorambucil at a dose of 40 mg/m² (83% versus 55%, p < 0.0001) and was associated with a 62% risk reduction in PFS. ⁷⁷⁰ This European trial justified a marketing indication for the use of alemtuzumab as initial therapy, but, in the United States, purine-analogue–based therapy is considered the treatment of choice, with or without cyclophosphamide, and is used typically in combination with rituximab.

Alemtuzumab is active against other CD52-positive lymphoproliferative disorders as well. As a single agent, alemtuzumab has produced RRs of 60% in cutaneous T-cell lymphoma (CTCL), ^{771 –774} 50% in prolymphocytic leukemia, ^775,776 and 40% in peripheral T-cell lymphoma (PTCL), ⁷⁷⁷ but only 20% in indolent B-cell lymphoma. ^{778 –780} Because these T-cell malignancies are relatively uncommon, and rituximab is more active in indolent B-cell lymphoma, most of the clinical use of alemtuzumab has continued to be in patients with CLL.

Because of additive toxicity and prolonged myelosuppression with the increased risk of opportunistic infections, combination biochemotherapy with alemtuzumab and chemotherapy has not been widely adopted. However, several trials have been conducted that have shown the feasibility of the approach in CLL ^781–782 and PTCL. ^783,784 There has been some exploration of the combination of rituximab and alemtuzumab as a combined immunotherapy approach, but RRs have been no better than rituximab with chemotherapy, and the combination treatment is more costly. ^{785 –787} A more-popular strategy has been to add alemtuzumab as a consolidation or maintenance therapy after an effective induction treatment for CLL. ^{788 –795} This approach has resulted in a higher rate of clinical, phenotypic, and molecular CR rates, and longer PFS, but there is an increased risk of severe opportunistic infections, even if antiviral, antibacterial, and anti–Pneumocystis carinii prophylaxis agents are used.

Trastuzumab

Trastuzumab (Herceptin®) is a humanized mAb that reacts with the human epidermal growth factor–2 (Her2). This mAB was granted regulatory approval in September 1998 for the treatment of breast cancer, thus becoming the the first mAb with approval based on activity in a solid tumor. ⁷⁹⁶ Trastuzumab is now part of standard treatment for Her2-positive breast cancer in both the adjuvant and metastatic settings. It is both a targeted therapy that interferes with tumor cell biology and an immunotherapy. ⁷⁹⁷ To prolong its half-life in the bloodstream and to promote immunotherapeutic effects, the idiotopes from the hypervariable region of 4D5, a murine mAb that binds Her2 (Her2/neu, c-ErbB-2) with high affinity and avidity, were joined with the elements of a human IgG₁ κ antibody using recombinant DNA technology. ⁷⁹⁸ Because of the human IgG₁ constant regions of the humanized mAb, trastuzumab is very effective for inducing ADCC in vitro against breast-cancer cells that overexpress Her2 when combined with human effecter cells. ^797,799 However, in vivo this may be limited by the internalization (downmodulation) of the Her2 target. Trastuzumab is ineffective for inducing CMC. Although interference with intracellular signal transduction is probably its most important antitumor effect, there is increasing indirect evidence that immune effects of trastuzumab are also important for its clinical activity. ^{800 –804}

Interference with intracellular signal transduction that facilitates cell proliferation is probably the most import antitumor effect of trastuzumab. Her2 is a member of the human epidermal growth factor receptor (EGFR) family that induces phosphorylation via tyrosine kinases. Interest in Her2 as a target followed the discovery that amplification of the erbB-2 proto-oncogene were associated with overexpression of the Her-2 receptor on tumor cells, and increased cell proliferation. ⁸⁰⁵ It was soon appreciated that ∼25% of breast-cancer patients had tumors that overexpressed Her2 and that this was associated with a worse prognosis. ⁸⁰⁶ Anti-Her2 mAbs competitively inhibit the ability of the receptor to dimerize with other members of the EGFR family and may also block their ligands, thus interfering with signal transduction that otherwise promotes cell proliferation. ⁷⁹⁷

As a single agent, trastuzumab has produced RRs ranging from 12% to 33% in patients with Her2-positive metastatic breast cancer. ^{807 –812} In the largest of these studies, a 15% RR was observed in 222 women whose cancers had recurred after chemotherapy, ⁸¹⁰ and a 26% RR was seen among 270 women treated with trastuzumab as initial therapy. ⁸¹⁰ In all trials, the best results with trastuzumab have been observed in patients whose tumors strongly overexpressed Her2 as seen on immunohistochemistry testing or on fluorescence in situ hybridization (FISH); therefore, accurate estimates of Her2 overexpression are important to justify such treatment. ^{812 –816}

The greatest benefits have been seen when trastuzumab has been combined with chemotherapy. Trastuzumab has been combined safely with a variety of single agents and used safely in combination chemotherapy regimens. ⁸¹⁷ Randomized trials have confirmed the superiority of adding trastuzumab to paclitaxel, ⁸¹⁸ docetaxel, ⁸¹⁹ and the combination of doxorubicin and cyclophosphamide ⁸¹⁸ as the initial chemotherapy for women with metastatic breast cancer. The benefits included higher RR, longer PFS, and longer OS. Trastuzumab has also been combined with a variety of other chemotherapy doublets utilizing taxanes and platinums. More recently, administration of trastuzumab concurrently, and/or following adjuvant chemotherapy, has been shown to prolong disease-free interval and OS in patients with node-positive and high-risk node negative disease. ^{820 –823} Trastuzumab has also been combined with chemotherapy in the presurgical setting. ^824,825 Although subsets of many other tumor types often overexpress Her2, most trials testing for added benefit for trastuzumab in those settings have generally been negative. However, recently a randomized trial confirmed that the addition of trastuzumab to chemotherapy improved survival for patients with adenocarcinoma of the gastroesophageal junction. ⁸²⁶

The most significant toxicity associated with trastuzumab use is cardiac dysfunction, or congestive heart failure, that typically is mild and reversible, ^827,828 but may leave behind permanent subclinical damage. ⁸²⁹ In the presence of clinical heart failure, or documentation of left-ventricular dysfunction, trastuzumab should be discontinued until cardiac function has improved. Trastuzumab can often be resumed without recurrence of left-ventricular dysfunction. During embryologic development, absence of the HER2/neu gene is associated with failure to develop a heart, ⁸³⁰ which suggests that the etiology for these cardiac effects may be trastuzumab targeting of Her2-positive cardiac muscle cells that are involved in tissue repair. As a single-agent, trastuzumab was associated with a 4%–5% rate of heart failure. ⁸⁰⁹ Clinical trials have shown that cardiotoxicity is markedly increased when trastuzumab is administered with or follows cardiotoxic agents such as anthracyclines, ^818,831 and therefore combining trastuzumab with such agents should be avoided. There has not been a direct comparison, but the risk of heart failure may be less for epirubicin than doxorubicin. ⁸³² Adjuvant trials utilizing doxorubicin and cyclophosphamide followed by paclitaxel or docetaxel have been associated with a heart-failure rate of 2%–4%. ^{833 –836} In an effort to avoid such risk, trastuzumab in combination with taxane-platinum doublets has gained in popularity. ^837,838 A randomized trial confirmed the superiority of carboplatin, paclitaxel, and trastuzumab, compared to paclitaxel and trastuzumab. ⁸³⁹ Such combinations are effective as adjuvant therapy in women with Her2-positive breast cancer.

Bevacizumab

Bevacizumab (Avastin®) is a humanized IgG₁ mAb that binds to and neutralizes the vascular endothelial growth factor (VEGF) ligand, rather than binding to a cell-membrane receptor. ⁸⁴⁰ This agent was originally approved in 2004 for use in combination with fluoropyrimidine-based chemotherapy in the first-line therapy of metastatic colorectal cancer, but subsequently has also received marketing indications in metastatic lung cancer, metastatic breast cancer, metastatic renal cell cancer, and glioblastoma. Based on the results of a recent randomized trial, bevacizumab will probably be granted a marketing indication in advanced ovarian cancer as well. ⁸⁴¹ Bevacizumab is a targeted therapy and is not believed to have relevant immunotherapeutic properties. VEGF, the central mediator of tumor angiogenesis, is produced by most types of malignant cells. A variety of in vitro and animal experiments established that VEGF would be a useful target for anticancer therapy. ^{842 –844} By blocking the ligand that activates the VEGF receptor on tumor blood vessels, bevacizumab potentially inhibits angiogenesis for any tumor type. It appears that bevacizumab treatment is also associated with afferent vascular dilatation and efferent vascular constriction of tumor vessels, which may help concentrate chemotherapy at the tumor site.

The most common adverse events associated with bevacizumab treatment are hypertension, proteinuria, hemorrhage, thromboembolic events, cardiac dysfunction, and impaired wound healing, and (rarely) perforated bowel. ^845,846 Protenuria can be severe and associated with nephritic syndrome. ⁸⁴⁷ Data from 7956 patients enrolled in 15 randomized trials identified a 12% risk of thromboembolic events in patients receiving bevacizumab, which was 33% higher than controls. ⁸⁴⁸ The impaired wound-healing has resulted in recommendations to wait 6–8 weeks after bevacizumab before performing elective major surgical procedures. ^849,850 Massive hemoptysis has been limited to lung cancer patients with large centrally located lesions that may be prone to cavitation. ⁸⁵¹ Although patients with brain metastases were excluded from bevacizumab trials in lung, colon, and breast cancer, because of concerns about the possibility of intracranial hemorrhage, such bleeding has not been a problem in patients with glioblastoma who were treated with bevacizumab. The rate of bowel perforation was 24/1442 (1.7%) at one large cancer institution. ⁸⁵² Bowel perforation has been most prevalent in colorectal and ovarian cancers that are prone to produce tumor implants in the bowel wall, but this complication has also occurred in patients who did not have bowel metastases. A DNA analysis of 363 breast cancer patients, who had been treated with bevacizumab and paclitaxel, found that VEGF-634 CC and VEGF-1498 TT genotypes were associated with significantly lower-grade severe hypertension. ⁸⁵³

Bevacizumab has shown limited antitumor activity as a monotherapy, with no responses recorded during phase I trials, ⁸⁵⁴ and RRs of 0% and 10% at two different doses in patients with renal cell cancer ⁸⁵⁵ and 3% in metastatic colorectal cancer. ⁸⁵⁶ Phase I studies established that bevacizumab could be combined safely with various chemotherapy agents. ⁸⁵⁷

In randomized trials, bevacizumab has consistently enhanced the antitumor effects of chemotherapy against established tumors. Combining bevacizumab with various chemotherapy regimens was better than the same chemotherapy alone in terms of RR, PFS, and OS in metastatic colorectal cancer, in both previously treated, ⁸⁵⁶ and previously untreated patients. ^{858 –866} However, a randomized trial in node-positive (stage III) colon cancer did not show a benefit for the addition of bevacizumab, compared to chemotherapy alone. ^867,868 Combining more than one mAb with chemotherapy has yielded surprisingly disappointing results in colorectal cancer. Combining cetuximab and bevacizumab with chemotherapy was associated with a worse outcome, compared to chemotherapy plus cetuximab. ⁸⁶⁹ Adding panitumumab to chemotherapy and bevacizumab was associated with a worse outcome, compared to chemotherapy plus bevacizumab. ⁸⁷⁰

In the first-line therapy of metastatic nonsquamous-cell lung cancer, becizumab increased the efficacy of taxane-platinum combinations in terms of RR, PFS, and OS. ^{871 –873} In heavily treated patients with metastatic breast cancer, the addition of bevacizumab to chemotherapy was associated with a doubling of the RR. ⁸⁷⁴ As a first-line therapy for metastatic breast cancer, adding bevacizumab to either paclitaxel or docetaxel taxane–based chemotherapy increased the RR and PFS, but not the OS. ^{875 –877} In metastatic renal cell cancer, as a single agent, bevacizumab had a 10% RR and was associated with longer PFS than placebo. ⁸⁷⁸ IFN-α plus bevacizumab produced a higher RR and PFS, compared to IFN-α plus placebo, ^194,195 but the difference in OS was not quite statistically significant, largely because of the effects of subsequent therapies at the time of disease progression. In patients with recurrent glioblastoma, the combination of bevacizumab and irinotecan were associated with ORs, ^879,880 but, subsequently, single-agent activity was established for bevacizumab as a monotherapy. ^881,882 The actual marketing indication is for monotherapy in the setting of glioblastoma. ⁸⁸³ Phase II trials suggested that bevacizumab might add to the efficacy of chemotherapy in ovarian cancer. ^884,885 A marketing indication in ovarian cancer is anticipated based on the results of randomized trial that was featured at the plenary session of the 2010 meeting of the American Society for Clinical Oncology. ⁸⁴¹ This was a 3-arm trial comparing chemotherapy alone to concurrent bevacizumab plus chemotherapy, and to concurrent bevacizumab plus chemotherapy followed by maintenance bevacizumab in patients with recently diagnosed advanced ovarian cancer that had been surgically debulked. The best results were seen in the third arm with a 38% risk reduction, compared to chemotherapy alone (p < 0.0001). One exception to these series of positive trials concerned advanced pancreatic cancer in which the addition of bevacizumab failed to improve on the benefits of gemcitabine alone, ⁸⁸⁶ or gemcitabine plus erlotonib. ⁸⁸⁷

Cetuximab

Cetuximab (Erbitux®) is an anti-EGFR chimeric mAb that was approved in 2004 based on randomized trials conducted in patients with colorectal cancer. ⁸⁸⁸ Like trastuzumab, cetuximab has some immunotherapy properties, but its antitumor effects are primarily mediated by interfering with signal-transduction pathways that are activated by the surface antigen. The anti-EGFR murine mAb C225 blocked EGFR-receptor function and inhibited the growth of human tumor cells in athymic mice. ⁸⁸⁹ Because the binding of C225 to the extracellular domain of EGFR resulted in internalization of the receptor and antibody, which limited CMC and ADCC, much of the early investigation with this anti-EGFR mAb focused on its potential conjugated to ricin to form an immunotoxin. ⁸⁹⁰ Cetuximab is a recombinant chimeric construct that includes a murine Fv and human IgG1 κ constant regions. The chimeric form of the mAb proved to be more effective than C225 in mouse tumor models. ⁸⁹¹ Unlike most mAbs that are currently approved for clinical use, cetuximab is manufactured in murine plasmacytoma cells.

The immunotherapy effects of cetuximab appear to be limited by rapid disappearance of the mAb after it binds to the EGFR receptor. ⁸⁹² Rapid disappearance of surface antigen after antibody binding apparently can occur by at least two processes. One is internalization inside the cell and the other is trogocytosis, a process in which effector cells remove and internalize ligands and cell-membrane fragments from the target cells. ^893,894 After internalization, once antibody is no longer present, cell antigens reappear, presumably because of recycling of the receptor. In vitro internalization of EGFR is slow enough that cetuximab is cytotoxic in the presence of human effector cells, but is less effective with complement. ⁸⁹⁵ Recent work focusing on Fc polymorphisms of effector cells provides indirect evidence that ADCC is a significant mechanism for the antitumor effects of cetuximab in vivo. ^896,897 However, the major mechanism of antitumor effects of cetuximab are its targeting of EGFR and disruption of cell-signaling pathways. ^888,898 EGFR is expressed in the vast majority of epithelial tumors and overexpression of EGFR is associated with increased cell proliferation, resistance to apoptosis, and increased VEGF expression. Binding of cetuximab to EGFR blocks phosphorylation and activation of several tyrosine kinases that are associated with the receptor, thereby disrupting signal transduction that is associated with increased cell proliferation and resistance to apoptosis.

As a single agent, in both single-arm, and randomized trials, cetuximab produced objective responses in 10%–15% of patients with metastatic colorectal cancer. ^{899 –903} Similar rates for single-agent activity were reported for head and neck cancer. ⁹⁰⁴ The RR was only 5% in previously treated metastatic non–small cell lung cancer (NSCLC), ⁹⁰⁵ and no acitivity was noted in metastatic renal cell carcinoma, ⁹⁰⁶ or ovarian cancer. ⁹⁰⁷

RRs and clinical benefit are much greater when cetuximab is combined with chemotherapy; thus, cetuximab is seldom used therapeutically as a single agent. Furthermore, adding cetuximab to chemotherapy provides benefit that is greater than with chemotherapy alone. In patients with metastatic colorectal cancer that had recurred despite prior irinotecan, RRs were twice as high for cetuximab and irinotecan, compared to cetuximab alone. ⁹⁰⁰ Randomized trials have confirmed that adding cetuximab to combination chemotherapy in the first-line treatment of patients with metastatic colorectal cancer is associated with higher RR and longer PFS, but only in patients with wild-type Kras, for both 5-FU-irinotecan, ⁹⁰⁸ and 5-FU-oxaloplatin based therapies. ⁹⁰⁹ Analysis of tumor specimens from patients with metastatic colorectal cancer who had enrolled in a randomized comparison of cetuximab versus supportive care showed the same predictive impact of the Kras mutation, but showed a lack of prognostic impact for the Kras mutation. ⁹¹⁰

It was somewhat surprising, and disappointing, that a randomized trial showed that adding cetuximab to the biotherapy combination of bevacizumab and chemotherapy resulted in a shorter PFS and inferior quality of life (QoL). ⁸⁶⁹ So far, a benefit for cetuximab has not been confirmed in other gastrointestinal (GI) malignancies. In a randomized phase II European trial that enolled 84 patients with locally advanced or metastatic pancreatic cancer, higher RRs were seen for the combination of cetuximab and gemcitabine versus the chemotherapy alone (17% versus 12, p = 0.055), ⁹¹¹ but, in a U.S., 745-patient randomized trial, there was no difference in RR or survival. ⁹¹²

Cetuximab appears to provide significant benefit in cancers of the lung and head and neck when added to chemotherapy and/or radiation therapy. In randomized trials in “wet” stage IIIB and stage IV NSCLC, adding cetuximab to carbolatin and a taxane resulted in a higher RR, but no significant improvement in PFS or OS in a U.S. trial that enrolled 676 patients. ⁹¹³ However, a survival advantage for cetuximab plus chemotherapy was confirmed in an European trial that enrolled 1125 patients. ⁹¹⁴ In 76 patients treated with cetuximab and paclitaxel plus carboplatin, patients whose cells had four or more gene copies per cell in >40% of the cells or gene amplification by FISH had longer PFS and OS. ⁹¹⁵ In contrast, in the U.S. randomized trial, there was no predictive correlation for outcome with either EGFR expression or for K-Ras mutation status. ⁹¹⁶ In the first-line treatment of patients with recurrent metastatic squamous-cell cancer of the head and neck, the addition of cetuximab to 5-FU and either cisplatin or carboplatin was associated with a higher RR, longer PFS, and longer OS. ⁹¹⁷ Adding cetuximab to radiation therapy also increased the survival of patients with measurable recurrent squamous-cell cancers of the head and neck. ^918,919

The major toxicities associated with cetuximab have been skin rash, diarrhea, and infusion reactions. In many trials, up to 80% of patients receiving cetuximab have developed a characteristic acneiform rash, which is most prominent on the upper body and face. ⁹²⁰ The rash usually appears within a week of starting treatment and is severe or life-threatening in 5%–15% of patients. Serial punch biopsies in patients revealed two main reaction patterns: a superficial dermal inflammatory cell infiltrate and a superficial folliculitis. The rash can be severe and should be treated to decrease the risk of secondary infection and cellulitis. Prophylactic topical lubricants are sufficient in many patients, but others require treatment with tetracycline antibiotics or clindamycin. Other common side-effects include asthenia and diarrhea. Cetuximab does not appear to increase the toxicity of any of the chemotherapy agents with which it has been combined.

Cetuximab infusions are occasionally associated with severe reactions that have included cardiac arrest and death, usually during the first exposure. ^921,922 There was a 2% frequency of sudden death in 208 patients with squamous-cell cancer of the head and neck who received cetuximab with radiation therapy. ⁹¹⁸ At this time, the etiology of such reactions is unclear and may be multifactorial. Cetuximab is the only mAb that is produced in a mouse plasmacytoma line, but this has not been implicated in the reactions to date. Wide regional variation in such reactions has been reported, and some patients apparently have a preexisting IgE that reacts with cetuximab. The frequency of reactions can be reduced by preadministration of corticosteroids in addition to antihistamines.

In summary, cetuximab is seldom used as a single agent but is commonly used in combination with chemotherapy in colorectal cancer and head and neck cancer. Targeted interference with signal transduction is considered to be this agent's major antitumor mechanism and the basis for synergy with chemotherapy rather than via immunotherapy mechanisms.

Panitumumab

Panitumumab (Vactibix®) became the first fully human mAb approved for cancer therapy when this agent received regulatory approval in September 2006. ^923,924 Panitumumab is a fully human IgG₂ that was originally made in transgenic mice but is manufactured in Chinese hamster ovary (CHO) cells. Like cetuximab, it binds to the extracellular domain of EGFR causing internalization of the receptor and antibody and disruption of downstream signal transduction that is associated with enhanced proliferation and resistance to apopotosis in normal and malignant cells. Panitumumab was specifically designed with the intent that it would be more active than cetuximab from an immunotherapy perspective because of greater CMC and ADCC. It was also hoped that the totally human construct would be associated with less toxicity. Like cetuximab, panitumumab reacts with EGFR-1 and was initially approved for metastatic colorectal cancer. In the case of panitumumab, it received regulatory approval on the basis of prolonging survival when used as a single agent in refractory metastatic colorectal cancer, compared to supportive care only. ⁹²⁵

In a 98-patient, dose-seeking, phase I trial panitumumab was well-tolerated and associated with an RR of 13% in 39 patients with metastatic colorectal cancer. ⁹²⁶ A multicenter, 84-patient phase trial found no significant differences in toxicity or efficacy for 6 mg/kg every 2 weeks, compared to 9 mg/kg every 3 weeks and also noted responses in patients with colorectal cancer. ⁹²⁷ In a Japanese trial, 7/52 (13%) of patients with colorectal cancer had a response. ⁹²⁸ In a phase II multicenter study, 148 metastatic colorectal cancer patients whose disease had progressed during or after treatment with a fluoropyrimidine and irinotecan, or oxaliplatin, or both, were treated with panitumumab monotherapy. ⁹²⁹ EGFR expression by immunohistochemistry of at least 1+ was required. Panitumumab was given i.v. at 2.5 mg/kg weekly for 8 of each 9 weeks until either disease progression or excessive toxicity occurred. The RR in this trial was 9%. Adverse dermatologic events occurred in 95% of patients and were severe or life-threatening for 5% of patients. Four (4) patients discontinued therapy because of toxicity. One (1) patient had an infusion reaction but was able to resume treatment. There was no difference in response for 105 patients who were judged as having high EGFR by IHC, compared to 43 patients who were characterized as having low EGFR. The RR for panitumumab monotherapy in metastatic colorectal cancer across these various trials is similar to what has been reported for cetuximab monotherapy.

Panitumumab was granted regulatory approval based on a randomized trial in which 6 mg/kg i.v. every 2 weeks was compared to best supportive care in patients with EGFR-positive metastatic colorectal cancer whose disease had progressed during or after standard therapy with fluoropyrimidine-, oxaliplatin-, and irinotecan-containing chemotherapy regimens. ^{925,930 –932} Panitumumab produced an RR of 10% and longer PFS, compared to supportive care alone. Perhaps because 75% of patients in the supportive care–alone arm ultimately received panitumumab after disease progression, there was no difference in OS. QoL was also better in the patients who received panitumumab. ⁹³³ The OR among the 176 patients who progressed on the supportive-care arm and then subsequently did receive panitumumab was 12%, including two CRs. ⁹³²

Because of the low level of clinical response, there has been interest in trying to detect markers that would predict benefit or lack of benefit from anti-EGFR therapies. The level of EGFR expression does not appear to be predictive. ⁹³⁴ However, similar to what has been reported for cetuximab, in colorectal cancer, it appears the presence of mutations B-Raf or K-Ras is associated with a lack of tumor response or clinical benefit from panitumumab. ^{935 –937}

Like cetuximab, panitumumab is being combined with chemotherapy in colorectal cancer, ⁹³⁸ and head and neck cancer. ⁹³⁹ In an important randomized trial of first-line therapy for metastatic colorectal cancer, the combination of chemotherapy plus bevacizumab and panitumumab was more toxic and associated with inferior PFS, compared to chemotherapy and bevacizuamb. ^870,940 At this time, no benefit for combining panitumumab with chemotherapy in the first-line treatment of metastatic colorectal cancer has been established. ⁹⁴¹

Generally, panitumumab has been well-tolerated whether it was administered alone or in combination with chemotherapy. The toxicities and adverse events associated with panitumumab are the same that have been observed with cetuximab, and include acneiform skin rash, diarrhea, and infusion reactions, and anaphylactoid reactions were reported for 1% of patients in the registration trial. Similar to other agents targeting the EGFR pathway, skin rash has been the primary toxicity recognized in association with panitumumab therapy and has occurred in 100% of patients receiving doses of 2.5 mg/kg or higher. ⁹⁴² Hypomagnesaemia and diarrhea were also most commonly reported. No grade severe or life-threatening reactions were noted in the large randomized trial in patients with colorectal cancer. ^931,932 Other common adverse events noted were paronychia, fatigue, abdominal pain, and nausea. The most serious adverse events were severe dermatologic toxicity complicated by infectious sequelae and septic death; infusion reactions; pulmonary fibrosis; hypomagnesemia; and GI problems, including abdominal pain, nausea, vomiting, diarrhea, and constipation. Despite being fully human, antipanitumumab immune responses were detected in 25 of 604 (4.1%) of patients enrolled in eight different clinical trials, including 8 patients who developed neutralizing antibodies. ⁹⁴³ At least 1 patient, who had a severe infusion reaction during treatment with cetuximab, was subsequently safely treated with panitumumab, ⁹⁴⁴ However, because most infusion reactions are seen only with a first infusion, the significance of this observation is unclear.

Panitumumab was the first fully human mAb to receive regulatory approval, and the second mAb approved that binds to the EGFR. At this time it is not clear whether the fully human construct is enhancing efficacy, especially in terms of CMC and ADCC.

Tremelimumab and ipilumumab

Although it had not received regulatory approval at the time of this review, it seemed highly probable that this mAb soon would receive a marketing indication for the treatment of metastatic melanoma. Ipilumumab is a fully human mAb that reacts with the cytotoxic T-lymphocyte antigen-4 (CTLA-4), a molecule that plays a critical role in the activation of T-regs that suppress immune responses and especially autoimmune responses. Preclinical trials showed that the CTLA-4 molecule reacted with the B7 family of costimulatory molecules in a manner that opposed the T-cell activating effects of interaction between B7 and CD28. ⁹⁴⁵ These effects included decreased T-cell proliferation and IL-2 secretion. In animal models, anti-CTLA-4 antibodies induced rejection of preestablished tumors. ^946,947 CTLA-4-deficient mice consistently developed a lethal polyclonal lymphoproliferative disorder. ⁹⁴⁸ These experiments established that CTLA-4 was a key regulator of T-cell responses and maintained the balance between CD4+ helper and CD8+ suppressor T-cells—especially Foxp3 + CD4+ regulatory T-cells. ⁹⁴⁹ Thus, CTLA-4 can inhibit T-cell activation and thereby maintain self-tolerance to antigens expressed on both normal and malignant cells. CTLA-4 is expressed on the surface of activated T-lymphocytes, where it suppresses the induction of immune responses that normally follow the interaction between T-cell receptors and HLA molecules on the antigen-presenting cells. Blocking CTLA-4 permits B7-CD28-mediated T-cell activation to go unopposed by B7-CTLA-4–mediated inhibition, which unleashes nonspecific autoimmune cytotoxicity.

Two fully human anti-CTLA-4 mAbs showed promising activity in early trials. ^950,951 These two products were tremelimumab (CP-675, 206; formerly known as ticilimumab), ^{952 –954} and ipilimumab (formerly known as MDX-010). ⁹⁵⁵ Melanoma appeared particularly appropriate for such therapy. ^{953,955 –957} The mechanism of an antitumor effect appeared to be nonspecific autoimmune inflammation, as both were associated with significant immune-related adverse events (IRAE), such as dermatitis, inflammatory bowel disease, uveitis, arthritis, hypophysitis, and others. Generally, these IRAE were reversible with cessation of therapy but typically required treatment with high doses of corticosteroids. The most frequent and significant toxicity was colitis and, unfortunately, partial or total colectomies were necessary in some severe cases of inflammatory bowel disease. ⁹⁵⁸ Unfortunately, several deaths occurred as a result of IRAE as well.

In the early trials of tremilimumab, a small percentage of ORs were documented, but many of these were durable and occurred in patients with advanced refractory disease. In a phase I dose-escalation trial of seven different doses in 39 patients, there were four responses among 29 melanoma patients. ⁹⁵⁹ Dose-limiting toxicities were IRAE, including autoimmune phenomena such as diarrhea, dermatitis, vitiligo, panhypopituitarism and hyperthyroidism, and uveitis. In a phase I/II trial in patients with metastatic melanoma, 28 went through the dose-escalation phase to determine a maximum tolerated dose, and then 8/84 responded to either 15 mg/kg i.v. every 3 months or 10mg/kg monthly. ⁹⁶⁰ There were four responses with each regimen, but the rate of grade 3 or 4 IRAE was twice as high for the monthly schedule. In a large phase II trial, 241 patients with metastatic melanoma received tremilimumab at a dose of 15 mg/kg i.v. every 90 days. ⁹⁶¹ The RR was only 7% and IRAE were significant with 11% of patients experiencing diarrhea and 1 patient dying. The median survival was 10 months, with 40% of patients alive after 1 year. The combination of tremelimumab and a vaccine consisting of dendritic cells pulsed with MART-1 was associated with objective responses in 4/16 patients. ⁹⁶² A phase II trial in metastatic colorectal cancer resulted in an OR in only 1 of 47 patients for tremelimumab at a dose of 15 mg/kg i.v. every 90 days. ⁹⁶³ Grade 3 and 4 IRAE included diarrhea (11%) and ulcerative colitis (2%). Based on the encouraging results in melanoma, a large phase III randomized trial was launched. However, in April 2008, Pfizer announced that it had discontinued the phase III clinical trial for metastatic melanoma patients after review of interim data showed that the tremilimumab arm could not possibly demonstrate superiority to standard chemotherapy. However, there was announced intent to pursue trials in prostate and bladder cancers.

Similar to the experience with tremilimumab, early trials of ipilumumab were associated with a small percentage of ORs, but those that did occur were impressive in terms of durability and the extent of disease in responders. Dose-escalation trials established that tumor regression could be induced in association with autoimmune toxicity. ^{964 –966} Encouraging effects were documented in several patients who had previously been treated with a vaccine and then subsequently received ipilumumab after tumor progression. ⁹⁶⁷ A 17% RR was observed in 139 metastatic melanoma patients who were treated with ipilimumab in conjunction with peptide vaccines. ⁹⁶⁸ IRAE were documented in 62% and included enterocolitis, arthritis, and uveitis. IRAE were highly correlated with RRs, and all patients who had a CR experienced grade 4 toxicities. The RRs were 9/24 among colitis patients and only 12/113 among patients who did not experience colitis (p = 0.006). ⁹⁵⁸ In a phase I/II trial in 58 patients with metastatic melanoma, one OR was noted, and 10mg/kg was considered to be the maximal tolerated dose. ⁹⁶⁹ In a randomized, placebo controlled trial that failed to show a benefit for the corticosteroid budesonide for decreasing the rate of enterocolitis, objective RRs of 12% and 16% were recorded for the two arms, with median survivals of 16 and 19 months. ⁹⁷⁰ In a 3-arm multicenter trial, 217 metastatic melanoma patients were randomized to one of three different doses of ipilumumab. ⁹⁷¹ The best response was 11% for the dose of 10 mg/kg and the percentage of patients experiencing IRAE was highest in this group.

For many years, there has been interest in using anti-CTLA-4 antibodies as an adjunct to vaccine therapy to inhibit regulatory T-cells that appear to inhibit the effects of such vaccines. For this reason, a randomized phase III trial was carried out on 676 HLA-A*0201-positive patients, 80% of whom were to receive the gp100 peptide based on a 3:1:1 randomization for ipilumumab plus gp 100, ipilumumab alone, and gp 100 alone. ⁵⁵⁵ These patients had unresectable stage III or IV disease and had experienced disease progression after having received at least one of the following agents: dacarbazine; IL-2; temoszolomide; fotemustine; or carboplatin. This randomized, double-blinded study was conducted in 125 centers in 13 countries with enrollment between September 2004 and August 2008. The study was originally designed based upon the endpoint of RR but was later modified for survival. The gp100 arm was clearly inferior to the two ipilumumab arms, which yielded similar results. The 1-year survival rates for the three arms were 44%, 46%, and 25%, respectively, and median survivals were 10.1, 10.0, and 6.4 months, respectively. Ipilimumab was associated with a 32%–34% decrease in the risk of death for the two arms. There were 14 deaths related to ipilumumab (2.1%). It is anticipated that this trial will probably lead to an accelerated approval of the product.

Other studies have suggested that ipilumumab produces activity in other cancers as well. In a phase II trial in metastatic renal cell cancer, PRs were declared for 6/61 patients (10%). Grades 3–4 toxicity were documented in 33% of patients. ⁹⁷² The RR was 35% among 17 patients who experienced colitis and only 2% among 44 patients who did not (p = 0.002). ⁹⁵⁸ Trials are ongoing in lung cancer and hormone-refractory prostate cancer and, no doubt, will be initiated in other malignancies as well.

In the modern era, one can anticipate the same explosion of combination trials as occurred with IFN-α and IL-2 because ipilumumab has the potential to augment a variety of vaccines and adoptive cell therapies on a purely immunotherapy basis. The biggest challenge for widespread use will be the significant toxicities that result from the autoimmune effects that ensue after suppression of T-regs.

Yttrium-90 ibritumomab tiuxetan

Yttrium-90 ibritumomab tiuxetan (Zevalin®) consists of the anti-CD20 murine mAb ibrubitumomab linked via the chelator tiuxetan to the Y-90 emitting radioisotope, which delivers ß radiation with a half-life of 2.7 days. ^{976 –978} In the United States, treatment is delivered by first administering indium-111 conjugated ibritumomab tiuxetan along with a low dose of rituximab as an imaging and blocking dose, followed by another low dose of rituximab and a Y-90 labeled mAb at what was determined to be the maximum tolerated dose with cytopenia being the dose-limiting toxicity. The pretreatment dose is not used for dosimetry purposes or to calculate the actual treatment dose. ⁹⁷⁹ Regulatory approval was granted in 2002 for treatment of indolent lymphoma. In the pre-rituximab era, after promising pilot studies, ⁹⁸⁰ clinical trials with the current product yielded objective RRs of 70% in phase I trials, ⁹⁸¹ 80% in phase II trials in follicular lymphoma after prior chemotherapy, ⁶³⁹ 70% in rituximab-refractory patients, ⁹⁸² 95% in previously untreated follicular lymphoma patients, ⁹⁸³ and 30% in patients with refractory mantle-cell lymphoma. ⁹⁸⁴ In randomized trials in indolent lymphoma patients, the radiolabled product produced a higher RR than standard dose rituximab. ⁶³⁹ A slightly lower dose was found to be equally effective in lymphoma patients with thrombocytopenia. ^985,986 Responses to radioimmunotherapy among patients with indolent lymphoma proved to be quite durable, with median TTP being almost 2.5 years. ^987,988 The RR was more than 80% in 35 indolent lymphoma patients who had recurred after prior rituximab alone or rituximab plus chemotherapy. ⁹⁸⁹

In a small study of 7 heavily treated patients with CLL or indolent lymphoma that had transformed to high-grade lymphoma, there were no responses and there was substantial hematologic toxicity. ⁹⁹⁰ Y-90 ibritumomab tiuxetan consolidation therapy was delivered to 13 patients with CLL, who were in complete or partial remission after induction therapy. ⁹⁹¹ Because of binding to circulating and bone-marrow CLL cells, high-grade toxicity was noted in 92%, although 1 patient did obtain a CR. Because of circulating CD20-positive cells and radiolabeled mAb distribution issues, such consolidation via the CD20 target should be reserved for patients with minimal residual disease.

Igs can penetrate into the brain when the blood–brain barrier has been disrupted. In a small pilot study of 6 patients with recurrent central nervous system (CNS) lymphoma, 10 × the dose of Y-90 was delivered to the tumor as to the normal brain. ⁹⁹² In a study of 9 patients with recurrent glioma, 3 had short-lived responses and 1 had a CR that persisted for more than 2.5 years. ⁹⁹³

While it is not practical to give RIT concurrently with chemotherapy because of overlapping hematopoietic toxicity, it is feasible to administer RIT as a consolidative therapy after chemotherapy has been completed. The addition of RIT increases CR rates and prolongs PFS. In a single-institution trial of 26 indolent lymphoma patients, 20 patients who had at least a PR following fludarabine and mitoxantrone chemotherapy and had normal platelets and <25% of the bone marrow infiltrated with malignant cells, the patients' therapy was consolidated with Y-90 ibritumomab tiuxetan, and 100% obtained a CR after RIT consolidation, although 75% experienced grade 3 or 4 hematologic toxicity. ⁹⁹⁴ In a multicenter trial utilizing a smiliar design in 61 patients with indolent lymphoma, 55/57 of patients obtained a CR after RIT, including 12/14 who had only had, a PR to induction chemotherapy. ⁹⁹⁵ Grade 3 or 4 hematologic toxicity was documented in 63%, and more than a third of the patients required red-cell transfusions. In 20 elderly patients with previously untreated diffuse large B-cell lymphoma, CHOP chemotherapy followed by Y-90 ibritumomab tiuxetan resulted in a 95% CR rate and 95% 2-year survival. ⁹⁹⁶ Four (4) of the 5 patients who did not have a CR after CHOP gained CR after RIT. While the early results of these Italian trials are excellent, their relevance is diminished because rituximab plus chemotherapy is standard initial therapy for patients with B-cell lymphoma rather than chemotherapy alone.

In a single-institution U.S. trial in 60 patients with follicular lymphoma, three cycles of RCHOP followed by Y-90 RIT, CR by positron-emission tomography (PET) scan improved from 46% to 86% in the 55 patients who completed therapy, and failure to achieve a CR by PET was associated with an increased risk of relapse within two years. ⁹⁹⁷ In a trial of 41 patients with follicular lymphoma, consolidation with Y-90 ibritumomab tiuxetan after rituximab with or without chemotherapy, was associated with an increased CR rate from 30% to 72% and a 64% 5-year PFS rate. ⁹⁹⁸ A 414-patient randomized trial in first-line therapy of follicular lymphoma confirmed that consolidation therapy with Y-90 ibritubomab tiuxetan increased the CR rate to 87% versus 53% in the control arm, and increased PFS from a median of 13 months to 36 months (p < 0.0001). ⁹⁹⁹ Unfortunately, only 14% of the patients had received rituximab as part of their induction therapy; so the value of such consolidation therapy following standard treatment is still unknown.

Higher doses of Y-90 ibritumumab tiuxetan have been given to patients in combination with autologous stem-cell rescue with the finding that hepatotoxicity becomes dose-limiting. ¹⁰⁰⁰ A 44-patient phase I/II trial has been reported for the combination of high-dose Y-90 ibritumomab tiuxetan plus BEAM (carmustine, etoposide, cyterabine, melphalan) followed by stem-cell rescue. ¹⁰⁰¹

Denileukin-diftitox

Denileukin-diftitox (Ontak®) is a genetically engineered fusion product that consists of IL-2 linked to the diphtheria endotoxin to produce the immunotoxin DAB₃₈₉IL-2. ¹⁰²⁴ The diphtheria toxin-receptor binding domain is replaced with IL-2 to target the IL-2 receptor. The immunotoxin is taken into cells via the high-affinity IL-2 receptor complex on CD25, where the immunotoxin is endocytosed into acidic vesicles, where the A chain toxin is released and inhibits protein synthesis that results in cell death within a matter of minutes. ¹⁰²⁵ A variety of lymphoid tumors, including CTCL, B-cell lymphoma, and Hodgkin's disease, express the high-affinity IL-2 receptor.

Phase I trials with a related product, DAB₄₈₆IL-2, established a maximum tolerated dose (MTD) on the basis of hypersensitivity-like symptoms and reversible hepatic toxicity manifested by elevated transaminases. ^1026,1027 One (1) patient with Hodgkin's disease had a CR. In a trial of 14 patients with CTCL, there was one PR, but 13/14 patients experienced transient trasaminasemia. ¹⁰²⁸

To create DAB₃₈₉IL-2 (denileukin diftitox), the second disulfide loop of the native diphtheria toxin was removed, which created a smaller and more biologically favorable fusion product that bound to the IL-2 receptor with greater affinity and was more potent and less toxic than DAB₄₈₆IL-2 in animal models. In a 73-patient phase I trial with DAB₃₈₉IL-2, in which asthenia was the dose-limiting toxicity, there was a 22% RR including 13/35 in patients with the mycosis fungoides stage of CTCL, 3/17 in B-cell lymphoma and 0/21 in Hodgkin's disease. ^1029,1030 A 30% RR was confirmed in a multicenter trial of 71 CTCL patients with one third of the responses being CRs. ¹⁰³¹ Combined safety data from these two trials indicated that 69% of patients experienced acute hypersensitivity-type reactions with 24 hours of treatment, 89% experienced flulike symptoms, 83% had hypoalbuminemia and an associated vascular-leak syndrome that resulted in clinical edema in 48%, and 50% had elevated hepatic transaminases. Based on these trials, denileukin diftitox was granted regulatory approval for the marketing indication of CTCL. Repeated treatment options are limited because of the immunogenicity of the diphtheria protein, which rapidly induces preexisting neutralizing antibodies and increased concentration in the liver and hepatotoxicity.

Additional trials have been conducted during the past decade since approval of denileukin diftitox. In 144 patients with CTCL, a 3-arm randomized trial compared two different doses of denileukin diftitox to placebo. ¹⁰³² This trial confirmed a 44% RR for the immunotoxin and 16% for placebo; so, to explain the high RR, it seems probable that patients were receiving other therapies as well. There are now data in PTCL in which a 48% RR was achieved in a 27-patient trial with a median PFS of 6 months. ¹⁰³³ As had been noted in several other trials, there was no significant difference in response on the basis of classification as being CD25+ or CD25−. In progressive B-cell lymphoma, a 24% RR was reported in 45 patients whose cancer had recurred after previous rituximab-based therapy, ¹⁰³⁴ but only a 10% RR was observed in a 29-patient trial, including 1/21 in follicular lymphoma and 2/8 in small lymphocytic lymphoma. ¹⁰³⁵ Again, there was no correlation between response and expression of CD25. The combination of denileukin diftitox and rituximab yielded an RR of 32% in 38 evaluable patients, 80% of whom were considered to be rituximab-refractory. ¹⁰³⁶ In recurrent CLL, objective RRs of 11% were documented in 18 patients whose disease was considered refractory to fludarabine, ¹⁰³⁷ and 27% in 22 patients with recurrent CLL. ¹⁰³⁸ Responses persisted for 2–20 months. In 41 patients with advanced NSCLC who had progressed after at least one prior treatment, there were no ORs. ¹⁰³⁹

From a purely immunotherapy perspective, denileukin diftitox is of interest because of its potential to perhaps preferentially deplete CD4+ CD25+ FoxP3+ T-reg cells that can suppress an endogenous or vaccine-induced immune response. Investigators have demonstrated that denileukin diftitox rapidly causes depletion of immune suppressing T regs. ^1040,1041 This is associated with the development of antigen-specific CD8+ cells in patients vaccinated with melanoma peptides. ¹⁰⁴⁰ However, based on the treatment of 12 patients with metastatic melanoma who had previously received IL-2, which is known to increase T-regs, ²⁷⁷ one group of researchers concluded that denileukin diftitox is not effective in reducing T-regs or inducing clinical remissions. ¹⁰⁴² In contrast, another group of researchers reported tumor regressions in 5/16 patients with metastatic melanoma and emergence of CD8+ cells specific for various melanoma antigens after treatment with denileukin diftitox. ¹⁰⁴³

Gemtuzumab ozogamicin

Gemtuzumab ozogamicin (Mylotarg®) consists of the humanized anti-CD33 monoclonal antibody gemtuzumab conjugated to colicheamicin, a semisythetic derivative of a potent antibiotic isolated from the Calichensis subspecies of the Micromonospora echoinospora. ¹⁰⁴⁴ It is estimated that about half of the immunoglobulin molecules remain unconjugated while the remaining half averages 4–6 molecules of toxin per immunoglobulin molecule. A human IgG3 backbone was used in an effort to limit interactions with immune-effector cells. Regulatory approval to market gemtuzumab ozogamicin for the treatment of relapsed acute myeloid leukemia (AML) was granted in May 2000, specifically for patients 60 or older who were not considered good candidates for chemotherapy. ^1044,1045 The approved dose was 9 mg/m2 i.v. over 4 hours repeated in 14 days.

In phase I trials the most common toxicities were the infusion reactions associated with the binding and clearance of circulating antigen-positive cells and myelosuppression. ¹⁰⁴⁴ CR were documented in 2/41 patients. Treatment was limited to two doses given at 2-week intervals because of immunogenicity and hematopoietic toxicity. Three multicenter, single-arm, phase II trials were conducted in patients with AML in first relapse, prior to regulatory approval. ¹⁰⁴⁵ The objective RR was 30% in 142 patients with a median age of 61. The median relapse-free survival for all 142 patients was 6.8 months. In addition to infusion reactions and myelosuppression, 23% had grade 3 or 4 hyperbilirubinemia, 17% had elevated hepatic transaminases, and 2 died with hepatic failure. Laboratory studies in 122 patients confirmed that the immunotoxin quickly bound to CD33+ cells and was rapidly internalized. ¹⁰⁴⁶ A follow-up summary on a total of 277 patients reported a CR rate of 26% and a PFS of less than 6 months, and confirmed the frequency and types of adverse events. ¹⁰⁴⁷ In subsequent trials, the single-agent activity of this immunotoxin was confirmed in a Japanese leukemia population. ¹⁰⁴⁸ The product also produced activity in acute promyelocytic leukemia. ¹⁰⁴⁹

Gemtuzumab ozogamicin was combined with a variety of AML regimens in various exploratory studies, but there did not appear to be any significant added benefit compared to chemotherapy alone. ^{1050 –1054} The feasibility of utilizing the immunotoxin as monotherapy for elderly patients who were not considered to be candidates for chemotherapy was demonstrated in small single-institution studies with a RR that was similar to that observed in the relapsed setting. ¹⁰⁵⁵ A 40-patient multicenter Italian trial of the immunotoxin as monotherapy had an RR of 70%, but the conventional 9 mg/m² dose was considered to be too toxic for patients over 75. ¹⁰⁵⁶ Lower doses appeared to be tolerated better with similar efficacy. ^1057,1058 A subsequent phase II randomized trial exploring different induction doses concluded that 6 mg/m² was safe and more active than 3 mg/m² in patients over 60 who were not considered candidates for cytotoxic chemotherapy. ¹⁰⁵⁹

Other trials examined gemtuzumab ozogamicin followed by the MICE regimen (mitoxantrone, cytarabine, etoposide) chemotherapy in patients ages 61–75. ¹⁰⁶⁰ Only 38/57 patients were able to complete the planned therapy but 31/38 went into remission (54% of the original 57 patients). Lethal toxicity was significant with three toxic deaths during immunotoxin therapy, five during MICE, and four from hepatotoxicity. In younger patients (ages 17–59) there was exploration of adding gemtuzumab ozogamicin with induction therapy and as consolidation in 72 patients. ¹⁰⁶¹ This proved to be difficult with substantial hepatotoxicity and prolonged cytopenias despite using 3 mg/m² and 6 mg/m² doses. In a randomized trial of 232 patients over 60, who had gained remission with induction chemotherapy, there was no benefit for the addition of the immunotoxin at 6 mg/m² as consolidation therapy. ¹⁰⁶²

The totality of these studies was disappointing in that gemtuzumab ozogamicin did not enhance any existing therapies. Also, there was increasing concern because of acute and delayed hepatotoxicity, especially in patients who subsequently underwent high-dose chemotherapy with stem-cell rescue, and in whom rates of veno-occlusive disease or sinusoidal obstructive syndrome were estimated to be between 10% and 40% if performed with 3 months of the immunotoxin therapy. ^1063,1064 Because randomized trials confirmed increased toxicity, but failed to confirm a survival advantage when the immunotoxin was used as a consolidation therapy after induction chemotherapy, the product was withdrawn from the market in the summer of 2010 at the request of the FDA.