Phase II Study of Alemtuzumab (CAMPATH-1) in Patients with HTLV-1–Associated Adult T-cell Leukemia/lymphoma

Adult T-cell leukemia/lymphoma (ATL) is an aggressive lymphoproliferative disorder occurring in individuals infected with the human T-cell leukemia virus type-1 (HTLV-1; refs. 1–3). ATL is characterized by the proliferation of malignant CD4⁺ CD25⁺ T cells in the peripheral blood, lymph nodes, and other tissues (3). The disease develops in 3% to 5% of HTLV-1–infected individuals after a latent period of 40 to 60 years (2, 4–6). ATL cells exhibit characteristic morphologic features (flower-like cells) with deeply indented nuclei, derived from mature regulatory T cells (Treg) with the surface phenotype (CD3⁺ dim, CD4⁺, CD7⁻, CD8⁻, and CD25⁺; refs. 1, 5). The aggressiveness of ATL varies and the disease has been grouped into 4 clinical subtypes: the “smoldering” subtype is characterized by at least 5% abnormal circulating T-lymphocytes with a normal peripheral blood lymphocyte count (4 × 10⁹/L), lack of hypercalcemia, serum lactic dehydrogenase (LDH) values no greater than 1.5 × the normal upper limit, and no lymphadenopathy or organ infiltration other than skin and lung; the chronic form of ATL manifests an absolute lymphocytosis (4 × 10⁹/L) and a T-cell lymphocytosis, elevated serum lactose dehydrogenase (LDH) values up to twice the upper limit of normal, without hypercalcemia or involvement of the central nervous system, bone, or gastrointestinal tract; the lymphoma subtype lacks a lymphocytosis and has less than or equal to 1% abnormal T cells in the circulation in conjunction with histologically proven malignant lymphadenopathy; and the acute type that includes patients with leukemic manifestations (7).

Despite significant advances in the understanding and treatment of many lymphoproliferative diseases, the prognosis for ATL remains poor with median survivals of 6.2, 10.2, and 24 months for acute, lymphomatous, and chronic subtypes, respectively (5, 8, 9). The majority of patients with the 3 more aggressive categories of ATL chronic, acute, and lymphomatous type have routinely been treated with anthracycline-based combination therapy including CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone). The Japanese Clinical Oncology Group demonstrated superiority of a complex multidrug chemotherapy regimen compared with biweekly CHOP in ATL in which the 1-year progression free survival rate was 28% (8, 9). Early relapse in ATL remains problematic despite any improvement in response rates or progression-free survival and the overall survival is poor. In the study by Gill, partial (6) or complete (5) remissions were observed in 11 of 19 patients with ATL treated with the combination of IFNα and zidovudine (AZT; ref. 10). However, the median survival of the whole group was only 3 months and 13 months for patients who obtained a complete or partial remission. A meta-analysis suggested that AZT therapy in combination with IFN may provide value (11). However, this combination has shown lack of efficacy in patients with prior chemotherapy who have p53 mutations or a high expression of IRF4 (12).

To develop an alternative to CHOP, we have turned to monoclonal antibodies directed toward receptors expressed on the surface of ATL cells. The development of therapeutic monoclonal antibodies has been a major advance in the treatment of B-cell lymphomas and other tumors. The introduction of rituximab (chimerized anti–CD-20) into the clinic has improved the response rate, progression-free survival, and overall survival of many patients with non–Hodgkin lymphoma of the B-cell linages, especially when combined with chemotherapy. We have evaluated receptor-directed monoclonal antibodies in a xenograft murine model of human ATL (13–15). Using the MET-1 in vivo model of ATL, we demonstrated efficacy with the CD2-directed monoclonal antibody, MEDI-507 (siplizumab), the anti-CD25 antibody, daclizumab, and the anti-CD52 monoclonal antibody, alemtuzumab (13–15). On the basis of these encouraging preclinical results, siplizumab, daclizumab, and now alemtuzumab (CAMPATH-1H) have been translated into clinical trials involving patients with T-cell malignancies with partial (PR) and complete responses (CR) observed (16).

Alemtuzumab is a humanized monoclonal antibody engineered by grafting the rodent hypervariable complementarity determining regions into a human immunoglobulin molecule (17). It is directed at CD52, a 12–amino acid peptide that is processed from a larger precursor, that is highly glycosylated and linked to the cell membrane by a phosphatidylinositolglycan linkage. CD52 is expressed on lymphocytes and monocytes, but monocytes and natural killer (NK) cells appear resistant to alemtuzumab-mediated lysis (18). It is estimated that there are 5 × 10⁵ antibody sites per lymphocyte and that the antigen does not modulate from the cell surface.

Examination of leukemic cells and fine-needle aspiration biopsies has demonstrated that CD52 is highly expressed on more than 90% of ATL cells, and its expression is comparable to the level of CD25 expression observed on the tumor cells in these patients. Binding of alemtuzumab to CD52 causes cell death through complement activation and antibody-dependent cell-mediated cytotoxicity (ADCC; ref. 19). As a result, alemtuzumab exhibits a powerful T-cell depleting effect and has been used to reduce the risk of graft-versus-host disease in patients undergoing allogeneic stem cell transplantation (20).

Alemtuzumab has demonstrated anticancer activity in patients with B-cell chronic lymphocytic leukemia (B-CLL; refs. 21–23), non–Hodgkin lymphoma (24, 25), T-cell prolymphocytic leukemia (PLL; refs. 26–28), mycosis fungoides, and Sézary syndrome (29–31). In a large number of patients studied with alemtuzumab by Wellcome, Inc. (32), they used an approach with 3 mg on day 1, 10 mg on day 2, 30 mg on day 3, followed by 30 mg intravenously 3 × weekly for up to 12 weeks. The incidence of common adverse events seen in about half of the treated patients included chills/rigors, fever, nausea, vomiting, and skin rash. Hypotension occurred in about a third of the patients. Dyspnea occurred in 25% and bronchospasm occurred in about 10% of patients. Thrombocytopenia occurred in association with the early infusions in the course of treatment. Tumor responses were observed in 16 of 174 patients. These responses occurred in patients with CLL or PLL leading to the approval of alemtuzumab for this indication. Alemtuzumab was granted initial approval by the FDA for intravenous use indicated as a single agent for the treatment of B-CLL in 2001. In 2007, the FDA granted approval for the use of alemtuzumab in previously treated patients with B-CLL after evaluation in an open-label: (i) active-controlled trial in previously untreated patients with B-CLL and (ii) Rai stage I–IV with evidence of progressive disease requiring therapy (33).

In preclinical studies, alemtuzumab prolonged the survival of mice with human MET-1 ATL comparable to that of tumor-free controls (14). These results were the basis of this single-institution, nonrandomized, open-label phase II clinical trial which was initiated to assess the antitumor activity and toxicity profile of alemtuzumab in patients with HTLV-1–associated ATL excluding those with the smoldering subtype.

To be included on the study, all patients had to have serum antibodies directed to HTLV-1 confirmed by Western blotting. A histologically confirmed diagnosis of ATL with more than 10% of the malignant cells expressing CD52 and CD25 as determined by flow cytometry or on immunohistochemical staining was required. CD25 (IL2Rα, Tac) expressing chronic, acute, and lymphomatous ATL subtypes were eligible. Patients with ATL were required to have measurable disease. Other requirements included age ≥ 18 years; absolute granulocyte count (AGC) ≥ 1,000/μL; platelet count ≥ 50,000/μL; serum creatinine ≤ 3.0 mg/dL, serum hepatic transaminases ≤ 2.5 fold greater than the upper limit of normal (ULN), total bilirubin ≤ 3.0 mg/dL, and a life expectancy of at least 2 months. Patients were required to discontinue all cytotoxic chemotherapy, zidovudine, biologic response modifier therapy, monoclonal antibodies, and other investigational antitumor agents at least 3 weeks prior to study entry.

This was a single-institution, nonrandomized, open-label phase II trial. Alemtuzumab was supplied through the Cancer Trials Evaluation Program (CTEP), NCI (Bethesda, MD). Alemtuzumab was administered as follows: on day 1, patients received alemtuzumab 3 mg, followed by 10 mg on day 2 and 30 mg on day 3, followed by alemtuzumab 30 mg 3 times per week, separated by 1 day (usually Monday, Wednesday, Friday, or Tuesday, Thursday, Saturday). A maximum of 12 weeks of alemtuzumab was administered. Patients also received antimicrobial prophylaxis with oral trimethoprim/sulfamethoxazole 160/800 mg 3 times per week and daily famciclovir or valganciclovir and fluconazole. Patients were switched to intravenous ganciclovir for increasing cytomegalovirus (CMV) antigen levels, or new onset antigenemia with signs of active infection.

CMV antigenemia in the first 7 patients was determined by quantitative immunofluorescence and in all subsequent patients by quantitative real-time PCR (qRT-PCR) performed on peripheral blood mononuclear cells (PMBC). PCR for detection of HTLV-1 and T-cell receptor (TCR) rearrangement was performed on DNA extracted from PBMCs by TaqMan assay (Applied Biosystems).

Bone marrow aspirate and biopsy were performed prior to treatment and every 4 weeks while on alemtuzumab in patients whose marrow was initially positive for tumor. Flow cytometry and lymphocyte subset analysis were performed on the peripheral blood at enrollment, and at weeks 4, 8, 12, 16, and all subsequent follow-up visits and included assessment of CD3, CD4, CD7, CD8, CD16, CD19, CD20, CD52, and CD25, the latter using the 7G7/B6 monoclonal antibody. After completion of the study, the patients were serially followed at least monthly if clinically indicated until the CD4 count recovered to greater than 200/mm³. In patients with leukemia, the PBMCs were analyzed for HTLV-1 integration and for clonal T-cell receptor gene rearrangements by PCR.

Drug safety was assessed by examining adverse events (AE), laboratory abnormalities, physical examination, and Karnofsky performance score status change from baseline (Table 3). Adverse events, serious adverse events (SAE) and abnormal laboratory values were graded using the CTEP Common Toxicity Criteria v 2.0. For monitoring, toxicity was defined over the 28-day period after treatment began. Patients who experienced grade 3 or 4 infusion-related AEs or who developed a grade 2 rash during the initial dose escalation had the lower dose of alemtuzumab continued until these toxicities resolved to less than or equal to grade 1. Alemtuzumab was discontinued in the event of grade 4 granulocytopenia persisting for more than 14 days despite growth factor therapy or grade 4 thrombocytopenia persisting for more than 14 days, grade 3 allergic reaction, grade 4 nonhematologic toxicity or grade 3 nonhematologic toxicity that did not resolve to less than grade 2 within 3 days with the exception of grade 3 infection.

Tumor response was determined using the International Workshop Standardized Response Criteria for non–Hodgkin lymphoma (34). Responses had to last for a minimum of 4 weeks to be determined as a response. Tumor status was evaluated at pretreatment, day 28, 56, and 72 on study and the date of study discontinuation. Patients achieving a CR at their evaluation time point or who had progressive disease (PD) defined as a persistent (at least 2 determinations) doubling of the peripheral blood leukemic cell count, the development of new lesions or serum calcium elevations that were uncontrolled by conventional therapeutic procedures at any time were removed from the study. Patients with a PR or stable disease (SD) received an additional 4 weeks of alemtuzumab therapy and were then similarly reevaluated. A maximum of 12 weeks of alemtuzumab were administered.

This was a single-arm, nonrandomized trial. An optimal 2-stage design (Simon 1989) to test for early evidence for futility based on CR and PR was used. The design was based on the following: (i) the response proportion (PR and CR) will be less than 5% if the treatment is totally ineffective (P₀ = 0.05), (ii) the treatment will be considered effective and worthy of future investigation if the true response proportion is consistent with 30% (P₁ = 0.30), (iii) a type I error rate of 5% (i.e., the probability of concluding that the treatment is effective if the true response rate is 5%, is 0.05), and (iv) a power of 95% (i.e., the probability of concluding that the treatment is effective if the true response proportion is 30%, is 0.95).

In the first stage of the 2-stage design, 9 patients were studied. An additional 20 patients (i.e., a total of 29 patients in the trial) were studied in the second stage. The major objective of the study was to determine the antitumor activity of alemtuzumab with regard to response rate, time to progression, and overall survival. Time to progression was measured from the date of registration until documentation of disease progression. Overall survival was determined from the date of registration to the event of death or for surviving patients, censored by the last day patients were known alive. The objective tumor response (CR + PR) rate and 95% confidence interval (CI) were calculated. For responders, the duration of response was measured from date of the response (CR + PR) to the documentation of disease progression or censored at the latest evaluation. Median duration of response was calculated using the Kaplan–Meier Method.

Between February 2004 and November 2009, 29 patients with HTLV-1–associated ATL were registered and treated with intravenously administered alemtuzumab on this trial at the NIH Clinical Center in Bethesda, MD (Tables 1 and 2). There were 19 women and 10 men with a median age of 48 years (age range, 24–76 years). Twenty-eight patients were Afro-Caribbean or African-American and 1 was Japanese. Eleven patients had lymphoma-type ATL, 15 had acute ATL, and 3 had chronic ATL. The median Karnofsky performance status was 89.4 (range, 80–90).

Twenty patients (69%) received prior treatment for their ATL, receiving a median of 1 prior regimen (range, 0–4). Prior chemotherapies included CHOP in 15 patients, zidovudine and IFNα in 1 patient, daclizumab (anti-CD25) monoclonal antibody in 3, and siplizumab (anti-CD2 monoclonal antibody) in 4 patients. One patient had received palliative radiation therapy. Nine patients were untreated prior to the trial. Six patients were receiving a stable dose of corticosteroids at the time of registration. Twenty-one patients (75%) completed treatment according to the protocol. The most common reason for early withdrawal was disease progression. The median time on the study was 6.7 weeks (range, 6–143.6 weeks), and the patients received a median of 1.6 cycles of alemtuzumab (range, 1–3 cycles). A cycle was defined as completion of 4 weeks of alemtuzumab at 30 mg 3 times weekly (total of 12 doses).

Grade 3 or higher infusion reactions were limited to 2 (7%) grade 3 vasovagal episodes and 3 (10%) hypotensive episodes (Table 3). All patients developed CMV antigenemia. Of these, 3 were symptomatic but all responded to antiviral therapy. Grade 3 or 4 infections were reported in 4 (14%) of the patients. Grade 3 and 4 adverse events occurring in 10% or more of the patients, outlined in Table 3, included hematologic (leukopenia: 41% grade 3 and 17% grade 4, lymphocytopenia: 59% grade 3, neutropenia: 31% grade 3 and 3% grade 4, anemia: 24% grade 3 and thrombocytopenia: 10% grade 3). After completion of the study, the patients were serially followed at least monthly if clinically indicated until the CD4 count recovered to greater than 200/mm³. The median time to recovery of absolute lymphocyte count (ALC) to > 200 cells/μL was 1.8 months.

The primary objective of this study was to determine the antitumor activity and toxicity profile of alemtuzumab in HTLV-1–associated ATL. The number of circulating cells expressing leukemic cell phenotype was monitored by direct and indirect immunocytofluoroscopy using a fluorescence-activated cell sorter (FACS). Fifteen (52%) of 29 patients with ATL responded to alemtuzumab, including 12 of 15 with acute ATL and 2 of 3 with chronic ATL (95% CI on 15 of 29, 32.5%–70.6%; Table 4). There was only 1 response among the 11 patients with lymphoma-type ATL. Six patients achieved a CR (21%) and 9 a PR (31%). For those who responded, the median time to response was 1.1 months (range, 1.0–4.2 months). Median duration of response was 1.4 months for the whole group and 14.5 months for responders. A Kaplan–Meier plot of overall survival is shown in Fig. 1; median survival was 5.9 months. A plot of survival for responders is shown in Supplementary Fig. S1. Median progression-free survival was 2.0 months and is shown in Supplementary Fig. S2.

There have been major advances in the therapy of aggressive B-cell lymphomas with the use of anthracycline-containing regimens such as CHOP and EPOCH with the anti-CD20 monoclonal antibody, rituximab. Therapeutic strategies with most forms of T-cell malignancies have been much less effective. By 2010, 4 agents (romidepsin, brentuximab vedotin, pralatrexate, and panobinostat) have been approved by the FDA for the treatment of various T-cell malignancies (35). However, when used as single agents, only about 30% of patients treated respond to therapy with a recurrence as the rule. A consensus has developed that multidrug combinations will be required for effective therapy of T-cell leukemia/lymphoma. Monoclonal antibodies have a number of features that support their inclusion in these multidrug combinations including excellent pharmacokinetics with a long survival duration in vivo, great specificity, and usually lack of bone marrow toxicity that would complicate therapy in association with chemotherapeutic agents (36). Phenotypic analyses of ATL cells revealed a high (>80%) incidence of expression of the chemokine receptor-4 (CCR4) as a hallmark of this disease (37, 38). Mogamulizumab (KW-0761) is a defucosylated humanized antibody with enhanced ADCC that binds to CCR4 and has yielded an overall response rate of 50% (in 13 of 26) in relapsed patients with CCR4-positive ATL (39, 40). Nevertheless, the anti-CCR4 monoclonal antibody is not curative and most of the patients relapse. As noted above, using the MET-I in vivo model of ATL, we demonstrated efficacy in mice with the anti-CD2 monoclonal antibody, siplizumab, the anti-CD25 antibody, daclizumab, and the anti-CD52 monoclonal antibody, alemtuzumab (CAMPATH-1; refs. 13–15). Furthermore, in our murine model, additive/synergistic responses were observed with the combination of daclizumab with bortezomib, flavopiridol, and romidepsin (41–43). In addition, in the MET-1 murine model, a synergistic response was observed with the combination of alemtuzumab and the putative survivin inhibitor, YM155 (44). Furthermore, we observed additivity/synergy when alemtuzumab was combined with 9AA, an agent that induces the activation of nonmutated but suppressed expression of p53 and activates NF-κB (45).

In the present study, we evaluated monoclonal antibody–mediated treatment in a phase II trial with patients receiving intravenous alemtuzumab 30 mg 3 times weekly for a maximum of 12 weeks. There was an overall objective response in 15 of 29 patients. Twelve of 15 patients with acute, 2 of 3 patients with chronic, and only 1 of 11 patients with lymphomatous HTLV-1–associated ATL manifested a response. However, the responses were brief with a median response duration of only 1.4 months for the whole group, 14.5 months among the 15 responders, and a median overall survival of only 5.9 months. These studies support further clinical trials in patients with chronic and acute forms of HTLV-1–associated ATL with alemtuzumab however in combination therapy.

One approach to be used with monoclonal antibodies such as alemtuzumab involves an effort to increase their efficacy by increasing their ADCC. ADCC involves the interaction of the Fc element of the monoclonal antibody with Fc alpha III and IV activating receptors on effector cells (46). In particular, in Fc receptor gamma^−/− mice, the efficacy of daclizumab and alemtuzumab in the MET-1 murine tumor model was lost (13, 14). A number of strategies have been used successfully to augment monoclonal antibody ADCC. These include defucosylation of the antibody or alteration of the Fc element to increase the binding of the monoclonal antibody to NK cells and monocytes that is required for its action (39). An additional approach involves the co-administration of an antibody to CD137 to activate NK cells—the cellular partner of the monoclonal antibody in ADCC (47). We are investigating yet another alternative, the use of IL15 in conjunction with alemtuzumab. The administration of tolerable concentrations of IL15 to mice, rhesus macaques, and to human patients has resulted in a 4- to 8-fold increase in the number of circulating activated NK cells (48). Furthermore, continuous intravenous infusion of IL15 at 20 μg/kg to rhesus macaques yielded a 15-fold increase in the number of circulating monocytes and an 80- to 100-fold increase in the number of circulating effector memory CD8 T cells (49). We have demonstrated a marked increase in the magnitude and duration of the antitumor response with the use of the anti-CD20 monoclonal antibody, rituximab, through ADCC with IL15 in a syngeneic model that involves EL4 cells transfected with human CD20 (50). Furthermore, in a syngeneic T-cell malignancy model using the cytokine-independent 43Tb⁻ ATL T-cell line in SCID/NOD mice, we demonstrated a similar increase in the duration of the antitumor response when alemtuzumab was used (50). On the basis of these studies, we have initiated a clinical trial that involves the evaluation of alemtuzumab in association with IL15 for patients with severe chronic and acute ATL (Clinical Trial NCT02689453).

In conclusion, in the present study, alemtuzumab induced responses in 12 of 15 patients with acute and 2 of 3 patients with chronic HTLV-1–associated ATL with acceptable toxicity but with a short duration of response. These studies support further clinical trials in patients with chronic and acute leukemic forms of HTLV-1–associated ATL with alemtuzumab given in conjunction with small-molecule agents that have shown efficacy in ATL or with IL15 to augment the ADCC action of the monoclonal antibody.

No potential conflicts of interest were disclosed.

Conception and design: J.E. Janik, J.C. Morris

Development of methodology: T. Fleisher, J.C. Morris

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): K. Sharma, J.E. Janik, D. O'Mahony, D. Stewart, S. Pittaluga, M. Stetler-Stevenson, E.S. Jaffe, M. Raffeld, T.A. Fleisher, C.C. Lee, T.A. Waldmann, J.C. Morris

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): K. Sharma, S. Pittaluga, M. Stetler-Stevenson, M. Raffeld, T. Fleisher, S.M. Steinberg, J.C. Morris

Writing, review, and/or revision of the manuscript: K. Sharma, J. Janik, D. O'Mahony, E.S. Jaffe, M. Raffeld, S.M. Steinberg, T.A. Waldmann, J.C. Morris

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J.C. Morris

Study supervision: J.E. Janik, J.C. Morris

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