Phase I Study of Inotuzumab Ozogamicin Combined with R-CVP for Relapsed/Refractory CD22+ B-cell Non-Hodgkin Lymphoma

Non-Hodgkin lymphoma (NHL) is estimated to be the sixth most common newly diagnosed cancer in the United States in 2015 (1). Although mAb-based therapies have advanced the treatment of many lymphoma subtypes (particularly B-cell NHL), relapse rates are high, and many patients are refractory to therapy (2–6). Combination treatments consisting of mAbs and chemotherapeutics have successfully treated relapsed/refractory NHL; however, toxicity remains a significant limitation with these regimens (7–9). Combination therapies that are both safe and effective are needed to meet the challenges of treating relapsed/refractory NHL.

The CD22 antigen is expressed by >90% of B-cell NHLs, is rapidly internalized, and is not shed into the extracellular environment (10–13). As such, it is an ideal target for mAb-based therapy for B-cell malignancies (10, 11). Inotuzumab ozogamicin (CMC-544) is a humanized immunoglobulin G4 anti-CD22 mAb conjugated to a derivative of calicheamicin, a potent cytotoxic antibiotic (12). Upon binding to inotuzumab ozogamicin, CD22 is rapidly internalized, releasing calicheamicin, which binds to DNA and induces double-strand DNA breaks, resulting in apoptosis (14–17). In contrast, the mechanism of action of the anti-CD20 mAb, rituximab, involves activation of immune system defense mechanisms, as well as directly inducing apoptosis (18). These disparate mechanisms of action allow for the possibility of synergistic effects between inotuzumab ozogamicin and rituximab; such effects have been observed in preclinical studies (19).

Previous phase I studies of inotuzumab ozogamicin demonstrated preliminary activity and manageable toxicity in patients with relapsed/refractory B-cell NHL (20–22). In addition, inotuzumab ozogamicin plus rituximab demonstrated preliminary activity in patients with relapsed/refractory B-cell NHL without a significant increase in toxicity (22, 23). Additive antitumor activity was observed in xenograft models with inotuzumab ozogamicin in combination with chemotherapeutics, such as cyclophosphamide, vincristine, and prednisone (CVP) (24). The alkylating agent–based immunotherapeutic regimen consisting of rituximab plus CVP (R-CVP) is approved for the treatment of previously untreated patients with stage III/IV follicular lymphoma in both Europe and in the United States (25). Treatment choice in relapsed patients depends on prior treatment history, type of lymphoma, and local treatment practices (26, 27). This phase I study determined the MTD and evaluated the safety and preliminary efficacy of inotuzumab ozogamicin given in combination with R-CVP in patients with relapsed/refractory CD22+ B-cell NHL.

Eligible patients were aged ≥18 years, with a diagnosis of CD22+ B-cell NHL (Table 1). Patients were required to have an Eastern Cooperative Oncology Group performance status of ≤2; total bilirubin ≤upper limit of normal (ULN; in patients without Gilbert disease); aspartate and alanine aminotransferase (AST and ALT) ≤2.5 × ULN; serum creatinine ≤2 × ULN and a urine protein/creatinine ratio of ≤0.5; an absolute neutrophil count ≥1.0 × 10⁹/L (1,000/μL) and a platelet count ≥100 × 10⁹/L; and ≥1 measurable disease lesion >1 cm in diameter (product diameter ≥2.25 cm² by CT or MRI). Patients in the dose-escalation cohorts had ≥1 prior anticancer treatment, including prior rituximab and chemotherapy. Enrollment of previously untreated patients in the MTD confirmation and preliminary efficacy cohorts was permitted if they were not considered candidates for anthracycline-based therapy.

Key exclusion criteria included the following: >3 previous chemotherapy regimens containing ≥2 cytotoxic agents; treatment with anti-CD22 antibodies, radioimmunotherapy or autologous transplant within 6 months of the first dose of study drugs; chemotherapy, cancer-immunosuppressive therapy, radiotherapy, growth factors (except for erythropoietin), or investigational agents within 28 days of first dose of study drugs. Also excluded were candidates for hematopoietic stem cell transplant; patients with a Fridericia corrected QT interval >470 msec at screening, symptomatic central nervous system disease, or a history of veno-occlusive disease/sinusoidal obstruction syndrome or chronic liver disease.

Written informed consent was obtained from each participant prior to study-related activities. The study (ClinicalTrials.gov: NCT01055496) was conducted in accordance with the International Conference on Harmonisation Guidelines for Good Clinical Practice and the Declaration of Helsinki. Institutional Review Boards and/or independent ethics committees at each participating center reviewed and approved the study protocol.

This open-label study was conducted at 12 centers in seven countries; patients were enrolled from March 2010 to February 2012. The study ascertained the safety, tolerability, and MTD (primary objective), as well as the preliminary efficacy and pharmacokinetics (secondary objectives) of inotuzumab ozogamicin given in combination with R-CVP in patients with relapsed/refractory CD22+ B-cell NHL. The study consisted of 3 parts: MTD determination (part 1), MTD confirmation (part 2), and MTD expansion (part 3). Patients received rituximab 375 mg/m² i.v., cyclophosphamide 375 to 750 mg/m² i.v., and vincristine 1.4 mg/m² (2 mg maximum) i.v. on day 1 plus prednisone 40 mg/m² on days 1 to 5, as well as inotuzumab ozogamicin 0.8 or 1.3 mg/m² i.v. on day 2 of each 21-day cycle (±2 days). Treatment continued for a maximum of 6 cycles unless there was evidence of progressive disease (PD) or prolonged or excessive toxicity; dose escalation was based on a toxicity assessment during cycle 1.

In part 1, dose escalation of cyclophosphamide (3 levels) or inotuzumab ozogamicin (2 levels) using a standard 3+3 design was used to determine the MTD (Supplementary Text and Supplementary Table S1). Initially, only the dose of cyclophosphamide was increased. Because the full dose of cyclophosphamide was tolerated, inotuzumab ozogamicin was increased to 1.3 mg/m². Ten additional patients were enrolled in part 2 to confirm the safety and tolerability of the MTD (cyclophosphamide 750 mg/m² and inotuzumab ozogamicin 0.8 mg/m²). Cohorts and treatment schedules are shown in Fig. 1. An additional 22 patients were enrolled in part 3. The preliminary safety and antitumor activity of inotuzumab ozogamicin plus R-CVP at the MTD was further examined in both parts 2 and 3.

Safety assessments included physical examination, vital signs, electrocardiograms, laboratory test measurements, and adverse event (AE) reporting. AEs were graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events version 3.0 and were monitored for ≥4 to 6 weeks after last dose (end-of-treatment visit). All treatment-related AEs were monitored until resolved, return to baseline, or stable condition. Disease assessments were performed until PD, initiation of a new anticancer therapy, or death; follow-up for survival continued for up to 2 years from the first dose of study drug.

Response and progression status were assessed using a modification of the International Working Group Criteria (28). Efficacy endpoints included the overall (objective) response [OR; complete response (CR) plus partial response (PR)], progression-free survival (PFS), overall survival (OS), and duration of response (DOR). Tumor responses were evaluated using objective measurements of tumor masses from MRI and CT scans, clinical information, B-symptoms, laboratory assessments, and biochemical markers of disease activity. Tumor site and measurements were determined at screening, every 9 weeks during the active period (or earlier if there was evidence of tumor response or progression), at the end-of-treatment visit, and then every 12 to 24 weeks until disease progression or new anticancer therapy or death for up to 2 years after the first dose of study drug.

Determination of pharmacokinetics included measurements of inotuzumab ozogamicin, total calicheamicin (conjugated plus unconjugated forms), and unconjugated calicheamicin concentrations in serum, which were collected during cycle 1 at 0 hour (predose), and during cycle 3 at 0 (predose), 1, 3, 24, and 144 hours after inotuzumab ozogamicin treatment. Serum samples to detect anti-inotuzumab ozogamicin and anti-rituximab antibodies were also obtained 24 hours predose during cycles 1, 4, and at the end of treatment. All analyses were performed using appropriately validated ELISA. For pharmacokinetic measures, analytes were quantified with limits of quantitation of 52.2, 5, and 1.25 ng/mL for inotuzumab ozogamicin, total calicheamicin, and unconjugated calicheamicin, respectively.

This preliminary study enrolled a limited number of subjects primarily to assess safety. The sample size was determined by clinical rather than statistical considerations. An analysis was performed for the 10 patients in the MTD confirmation cohort, and the decision to proceed to part 3 was based on information available from the unlocked database at the time of this analysis. Continuation of the study to part 3 required a CR or PR in ≥2 patients, <33% of patients experiencing a dose-limiting toxicity (DLT), and <33% unable to receive ≥3 cycles due to a drug-related toxicity. Outcomes are reported on the basis of final data for all treated patients. PFS, OS, and DOR distributions were summarized using the Kaplan–Meier method.

Forty-eight patients (indolent, n = 27; aggressive, n = 21) were enrolled in the study (Fig. 1); baseline patient demographics and disease characteristics are presented in Table 1. Patients had follicular lymphoma (54%), diffuse large B-cell lymphoma (DLBCL; 35%), mantle cell lymphoma (8%), or small lymphocytic leukemia (2%) at screening and had received a median of two prior therapies (range, 1–6). All patients received prior anticancer therapy that included rituximab except for one patient enrolled in part 3. Eight patients (indolent, n = 3; aggressive, n = 5) had refractory disease at baseline (best response to most recent prior therapy was stable or progressive disease).

All patients received ≥1 dose of study treatment and most (52% total; 50% at the MTD) completed all six treatment cycles with a median (range) of six (1–6) cycles completed (one patient did not receive vincristine during the last two treatment cycles). Reasons for discontinuing treatment included AEs [n = 13 (27%)], PD [n = 9 (19%)], patient request [n = 2 (4%)], and death [n = 2 (4%)].

In part 1 (dose escalation), the MTD was determined to be inotuzumab ozogamicin 0.8 mg/m² given on day 2 with standard-dose R-CVP (rituximab 375 mg/m², cyclophosphamide 750 mg/m², and vincristine 1.4 mg/m² all on day 1, and prednisone 40 mg/m² on days 1–5) intravenously every 21 days (29). DLTs observed among the 16 patients treated in part 1 are summarized in Fig. 1. No DLTs were reported with inotuzumab ozogamicin 0.8 mg/m² at the lower doses of cyclophosphamide (levels 1 and 2; n = 3 each). One of six patients at dose level 3 experienced a DLT (grade 4 neutropenia requiring G-CSF); two of four patients reported DLTs at dose level 4 (one patient had grade 4 neutropenia requiring G-CSF; one had grade 4 thrombocytopenia and grade 2 acute hepatitis lasting ≥7 days).

The MTD was confirmed in part 2. Two of 10 patients had DLTs including grade 3 increase in ALT (recovered after 8 days) and AST (recovered after 6 days) in one patient, and grade 4 neutropenia requiring G-CSF. One patient discontinued treatment due to AEs (grade 2 thrombocytopenia and grade 3 neutropenia, both treatment related) prior to cycle 3. Seven of 10 patients received ≥3 cycles of study treatment, and ≥5 patients had at least a PR as a best overall response; therefore, investigators were instructed to proceed to part 3.

Among 38 patients treated at the MTD, the most common treatment-related AEs of any grade were hematologic abnormalities, fatigue, liver enzyme abnormalities, and gastrointestinal toxicities; hematologic abnormalities accounted for most grade ≥3 treatment-related AEs (Table 2). Twenty-one patients treated at the MTD had reported platelet laboratory values corresponding to grade ≥3 decreased platelets, 18 of whom recovered to grade ≤1 while on study after a median of 14 (range, 6–474) days. Twenty-seven patients treated at the MTD had reported neutrophil laboratory values corresponding to grade ≥3 neutrophil count decreased, all of whom recovered to grade ≤1 after a median of 9 (3–181) days. Three patients receiving the MTD experienced treatment-related QT prolongation (grade 1, n = 1; grade 2, n = 2).

A total of 13 patients permanently discontinued treatment due to AEs, 11 of whom were treated at the MTD (Supplementary Table S2). AEs leading to discontinuation of treatment at the MTD were thrombocytopenia or delayed (>28 days) recovery from thrombocytopenia [n = 9 (grade 2, n = 6; grade 3, n = 1; grade 4, n = 2)], grade 3 neutropenia (n = 1; together with grade 2 thrombocytopenia), grade 2 ALT increased that prevented redosing for >28 days (n = 1), as well as grade 1 alkaline phosphatase and ALT increased (n = 1; patient had no subsequent laboratory assessments).

Six patients treated at the MTD (eight patients total) had AEs leading to dose reductions, including neutropenia (n = 2), leukopenia, thrombocytopenia, ALT increased, AST increased, weight decreased, and fatigue (n = 1 each; Supplementary Table S2). AEs leading to dose delays occurred with 24 patients receiving the MTD (26 patients total). Of these AEs, only neutropenia and thrombocytopenia (n = 10 each) occurred in >2 patients.

A total of 13 deaths were reported, most commonly due to PD (n = 9). Only one patient died within 28 days after the last dose of study treatment (due to cardiopulmonary arrest secondary to DLBCL). There was one report of treatment-related fatal pneumonia associated with grade 4 neutropenia; other reasons for death include cardiorespiratory arrest and lung cancer (n = 1 each).

Among the 38 patients treated at the MTD, the overall response rate (ORR) was 84% [95% confidence interval (CI), 69%–94%], including nine CRs (Table 3). All patients treated at lower levels responded [dose level 1 (n = 3), two CRs and one PR; dose level 2 (n = 3), one CR and two PRs]; of the four patients treated at level 4, one had a CR. All 27 patients with indolent B-cell NHL responded, including 11 (41%) who achieved a CR. Among the 21 patients with aggressive B-cell NHL, a response was observed in 12 (57%) patients, two of whom achieved a CR. In addition, a response of CR or PR was achieved by five of eight patients who were refractory to most recent prior therapy, including two with aggressive histology lymphoma.

Kaplan–Meier estimated probabilities of PFS, OS, and DOR among all patients (n = 48) and those with indolent (n = 27) or aggressive (n = 21) B-cell NHL are shown in Fig. 2. A total of 28 patients experienced PFS events (PD, death, or new anticancer therapy). Among all 48 patients, the median (95% CI) PFS was 14.4 [6.9–not yet reached (NR)] months, and Kaplan–Meier estimated probability (95% CI) of PFS at 24 months was 0.38 (0.23–0.52). With 13 and 15 events, respectively, the median PFS was not reached for all patients with indolent B-cell NHL and was 4.4 (1.3–12.9) months for those with aggressive B-cell NHL. The Kaplan–Meier estimated probability (95% CI) of PFS at 24 months was 0.50 (0.29–0.68) for all patients with indolent B-cell NHL and 0.19 (0.05–0.40) for all patients with aggressive B-cell NHL.

After a median follow-up of 24.1 (range 1.3–38.1) months, 22 patients receiving the MTD had experienced PFS events. Among all 38 patients treated at the MTD, the median (95% CI) PFS was 14.4 (5.5–NR) months. The Kaplan–Meier estimated probability (95% CI) of PFS at 24 months was 0.39 (0.23–0.54; Table 3). For the 18 patients with aggressive B-cell NHL receiving the MTD, the median (95% CI) PFS was 4.4 (2.4–14.4) months, and Kaplan–Meier estimated probability (95% CI) of PFS at 24 months was 0.21 (0.06–0.44).

As of the data cutoff date (October 2, 2014), the median OS has not been reached for patients who received the MTD. The median (95% CI) OS was also not reached for patients with indolent B-cell NHL and was 24.5 (5.5–NR) months for patients with aggressive B-cell NHL. The Kaplan–Meier estimated probability (95% CI) of OS at 24 months was 0.74 (0.56–0.85) for patients receiving the MTD, 0.89 (0.69–0.96) for patients with indolent B-cell NHL, and 0.55 (0.31–0.73) for patients with aggressive B-cell NHL.

Among 32 responders treated at the MTD, the median (95% CI) DOR was 21.4 (9.2–NR) months (Table 3). Response duration was longer for patients with indolent [NR (11.2 months–NR)] versus aggressive [11.0 (1.8–NR) months] B-cell NHL.

Pharmacokinetic data from the start of cycle 3 were available from 23 patients treated at the MTD. Mean concentrations of inotuzumab ozogamicin and calicheamicin during cycle 3 are shown in Table 4. The concentrations of unconjugated calicheamicin were predominantly below the limit of quantification. Data for inotuzumab ozogamicin and total calicheamicin indicate that peak exposures were achieved at or near the end-of-infusion time, with high exposures minimally maintained through 24 hours. Total calicheamicin concentrations were usually detectable for approximately 1 week after dosing.

Immunogenicity testing using ELISA indicated that 5.9% (7/118) of samples in 8.5% (4/47) of patients were positive for anti-inotuzumab ozogamicin antibodies; however, these positive samples were primarily obtained before cycle 1 and were not considered related to inotuzumab ozogamicin treatment. Antibodies to inotuzumab ozogamicin were detected in two patients after inotuzumab ozogamicin treatment, neither of whom exhibited clinical symptoms attributable to the formation of anti-inotuzumab ozogamicin antibodies. No anti-rituximab antibodies were detected among 116 samples drawn from 46 patients.

Currently, there is considerable interest in the potential for delivering targeted cytotoxic chemotherapeutic agents for the treatment of lymphomas by conjugating these agents with specific mAbs that recognize highly expressed cell surface proteins (30–32). Despite the progress in initial treatment of B-cell NHL using combinations of mAbs with chemotherapy, there remains an unmet clinical need as most adult patients with NHL relapse, and many become refractory to standard therapy. Results from in vitro studies suggest mAbs can sensitize lymphoma cells to chemotherapy (33, 34), and a synergistic effect was observed between rituximab and various cytotoxic agents in patients with relapsed/refractory disease (35–39). In addition, unconjugated mAbs generally have mild, nonoverlapping toxicities with most cytotoxic agents (40). We therefore assessed the safety, MTD, and preliminary efficacy of the addition of another targeted agent (inotuzumab ozogamicin) to R-CVP in patients with relapsed/refractory CD22+ B-cell NHL.

The MTD for inotuzumab ozogamicin plus R-CVP was determined to be inotuzumab ozogamicin 0.8 mg/m² plus standard-dose R-CVP given on a 21-day cycle; DLTs were hematologic (grade 4 neutropenia and thrombocytopenia) and hepatic (grade 2 hepatitis). Although dose escalation of cyclophosphamide in this combination was possible, the inotuzumab ozogamicin dose could not be increased. Consistent with the observed DLTs, toxicities associated with inotuzumab ozogamicin plus R-CVP were frequently hematologic [most commonly thrombocytopenia (83%)]; these toxicities were also the most common reason for treatment discontinuations, dose delays, and/or dose reductions. However, most patients had received ≥2 prior therapies, potentially predisposing them to developing hematologic AEs. Whereas fatigue (50%), constipation (42%), and nausea (47%) were the most common (any grade) treatment-related nonhematologic AEs in patients treated at the MTD, liver test abnormalities, including ALT increased (32%), AST increased (37%), and hyperbilirubinemia (34%) were also frequent and were a common reason for treatment discontinuation or dose reduction. Grade 3/4 nonhematologic AEs were uncommon. Overall, the toxicity profile of this combination was consistent with that previously reported for inotuzumab ozogamicin alone (18, 19), or in combination with rituximab (20, 21).

The benefit of adding rituximab to chemotherapeutic regimens to response and survival rates is well established (2, 3, 41–45), and immunochemotherapies have become standard treatments for relapsed/refractory disease. However, the addition of anthracyclines to CVP significantly increases the toxicity of the regimen (46, 47); R-CVP may be preferable to anthracycline-containing regimens in patients with a known cardiac history or for whom cumulative cardiac toxicity may be of concern; as such, R-CVP was chosen in this study for coadministration with inotuzumab ozogamicin.

Interpretation of the efficacy data is limited by the small sample size and heterogeneity of enrolled patients. In addition, observed responses and response durations should be interpreted in the light of the low risk of many of the patients enrolled in this study, as 13 (34%) of the 38 patients treated at the MTD had stage I/II disease at study entry and 16 (42%) had achieved a CR to their most recent prior regimen. Nevertheless, the preliminary efficacy is encouraging, with 11 and two CRs observed in patients with indolent and aggressive B-cell NHL, respectively. The ORR for all follicular lymphoma patients receiving inotuzumab ozogamicin plus R-CVP (100%) compares favorably with that reported for frontline R-CVP in patients with follicular lymphoma (81%; ref. 44) and appears similar to that reported for rituximab plus inotuzumab ozogamicin (80%) in early-phase studies (22, 23). In addition, there was a 57% response rate in patients with aggressive lymphoma. This suggests that it may be appropriate to examine inotuzumab ozogamicin in combination with R-CVP in patients unable to tolerate anthracyclines (48), and a study of this regimen in chemotherapy-naïve patients with DLBCL who are not candidates for anthracycline-based treatment is currently recruiting (49). Treatment at the MTD was also associated with notable PFS and OS rates at 24 months of 39% and 74%, respectively.

Circulating levels of inotuzumab ozogamicin, total calicheamicin, and unconjugated calicheamicin appear comparable with those previously reported following concomitant treatment of inotuzumab ozogamicin with rituximab alone in patients with B-NHL (23). The potential for the formation of anti-inotuzumab ozogamicin and anti-rituximab antibodies appears low.

In conclusion, inotuzumab ozogamicin 0.8 mg/m² given with standard-dose R-CVP demonstrated manageable safety and tolerability. Nearly all grade 3/4 AEs in this heavily pretreated population were hematologic, most commonly thrombocytopenia and neutropenia. Although the antitumor activity of this combination was noteworthy, further investigation in a larger population would be required to determine whether this regimen offers an advantage over other previously established regimens or whether inotuzumab ozogamicin should be combined with other agents.

M. Ogura reports receiving speakers bureau honoraria from Takeda Pharmaceutical Co., Ltd., and is a consultant/advisory board member for Celgene, MeijiSeika Pharma, Mundipharma, and Sandoz. K. Tobinai reports receiving commercial research grants from Abbvie, Celgene, Chugai Pharmaceutical, Eisai, GlaxoSmithKline, Janssen Pharmaceutical, Ono Pharmaceutical, and Servier. A. Davies is a consultant/advisory board member for CTI, Gilead, Karyopharma, Mudipharma, Roche, and Takeda, reports receiving commercial research grants from Acerta, Bayer, Gilead, GlaxoSmithKline, Karyopharma, Pfizer, Roche, and Takeda, and reports receiving fees for presentation at an educational meeting from Janssen. M. Crump is a consultant/advisory board member for Celgene, Roche Canada, and Seattle Genetics. L. Paccagnella and E. Vandendries have ownership interest (including patents) in Pfizer. D. MacDonald is a consultant/advisory board member for Roche Canada.

Conception and design: J. Boni, E. Vandendries

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): M. Ogura, K. Tobinai, K. Hatake, A. Davies, M. Crump, D. MacDonald

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): M. Ogura, K. Tobinai, A. Davies, M. Crump, R. Ananthakrishnan, T. Ishibashi, L. Paccagnella, J. Boni, E. Vandendries

Writing, review, and/or revision of the manuscript: M. Ogura, K. Tobinai, A. Davies, M. Crump, R. Ananthakrishnan, T. Ishibashi, L. Paccagnella, J. Boni, E. Vandendries, D. MacDonald

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): T. Ishibashi, L. Paccagnella

Study supervision: L. Paccagnella

We would like to thank the patients who participated in this study and their families, as well as the research nurses, study coordinators, and operations staff. This study was sponsored by Pfizer Inc. Editorial support was provided by Johna VanStelten, PhD, and Simon J. Slater, PhD, of Complete Healthcare Communications, LLC., and was funded by Pfizer Inc.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.