Phase I Safety and Pharmacokinetic Study of CT-011, a Humanized Antibody Interacting with PD-1, in Patients with Advanced Hematologic Malignancies

Patients and Methods

CT-011 mAb. CT-011 has been produced and provided for this clinical trial by CureTech Ltd.

Patient selection. Entrance criteria for the study required that patients enrolled in the study had to have one of the following hematologic malignancies: acute myeloid leukemia (AML), chronic lymphocytic leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, or multiple myeloma at an advanced stage of their disease and following chemotherapy and/or stem cell transplantation. Patients were eligible for this study provided that they met the following criteria: age 18 to 65 y; measurable disease (blast count and morphology, computed tomography scan or protein electrophoresis, β₂ microglobulin, immunoelectrophoresis, Bence Jones protein in urine); at least 4 wk from stem cell transplantation or 1 wk from donor lymphocyte infusion; Eastern Oncology Cooperative Group (ECOG) performance score <2; life expectancy >3 mo; hematology: WBC >0.5 × 10⁹/L, hemoglobin >7 g/dL, and platelet count >10 × 10⁹/L; adequate renal function: creatinine <2.0 mg/dL; adequate hepatic functions: bilirubin, aspartate aminotransferase, or alanine aminotransferase <3 times the upper limit of reference ranges; normal cardiac function: no prior coronary artery disease; no New York Heart Association class II, III, or IV congestive heart failure; and no arrhythmia requiring treatment. Patients who were either receiving or did not recover from the effect of therapies having immune suppressive effects or who were suffering from an autoimmune disorder were to be excluded. The exception to this was hydroxyurea treatment of AML patients, which was allowed to proceed. All patients gave written informed consent before study entry. The study was approved by the Medical Ethics Committee of the institution and the Israeli Ministry of Health. Evaluation of the disease status as well as overall medical condition was conducted before CT-011 treatment. A complete medical history was recorded and a physical examination done. Blood tests including complete blood count and serum biochemistry were conducted.

Treatment and dose escalation. This was a phase I, nonrandomized, open-label, safety study of escalating doses of the mAb CT-011 administered as a single i.v. infusion. CT-011 (humanized mAb) for parenteral administration was supplied by CureTech. A total of 15 patients were planned to be enrolled in the study with cohorts of 3 patients in each of five dose levels of 0.2, 0.6, 1.5, 3, and 6 mg/kg. The study enrolled a total of 17 patients. One patient who was enrolled at the lowest dose level (0.2 mg/kg) was re-enrolled 5 mo after the first administration at a higher dose level (3.0 mg/kg) as a compassionate treatment, for a total of 18 administered treatments. The total amount of CT-011 was determined based on the planned dosing (mg/kg base) and body weight. To control for any infusion-related symptoms, the infusion was carried out in a stepwise manner, increasing the rate from 50 to 100 mL/h. In addition, as customary for several marketed mAbs, all patients that participated in the study received premedication before infusion, consisting of a pain relief medication (paracetamol), corticosteroid (hydrocortisone 100 mg), and an antihistamine (phenergan). The starting dose for this study was 0.2 mg/kg, which was several 10-fold lower than the highest dose tested in toxicology studies conducted in nonhuman primates and mice on a human-equivalent-dose base. The further dose levels were 0.6, 1.5, 3, and 6 mg/kg. Escalation from one dose level to the next was allowed after all patients at the previous level were evaluated for at least 7 d following the dose administration. Infusion of CT-011 was done by hospital personnel. Patients were then to be hospitalized and monitored for 24 h from the commencement of the infusion.

The use of concomitant anticancer treatment (chemotherapy and immunotherapy) was prohibited and accordingly was to be stopped at least 4 wk before CT-011 administration. The exception to this was hydroxyurea treatment of AML patients, which could proceed. Given the mechanism of action of CT-011, the use of chronic treatment that induces immune suppression was prohibited (e.g., chronic treatment with corticosteroids), but an acute treatment was allowed. Consequently, cyclosporine A treatment was to be stopped 1 wk before CT-011 administration. Otherwise, all medications that were clinically indicated as well as medications that had previously been taken for conditions other than the malignant disorder were allowed and were recorded.

Safety assessment. The primary objective of the study was to determine the safety, dose-limiting toxicity, and maximal tolerated dose of a single i.v. administration of CT-011 to patients with advanced stage hematologic malignancies. Toxicity was evaluated according to the National Cancer Institute Common Toxicity Criteria for Adverse Events version 2 and by its intensity (i.e., mild, moderate, severe). Escalation from one dose level to the next was to be determined after all patients at the previous level were evaluated for at least 7 d following the dose administration. Dose-limiting toxicity was defined as that dose which induces any grade 3 or 4 toxicity in one or more patients, or any grade 2 toxicity at least in 2 of 3 or in 3 of 6 patients. If a grade 2 CT-011-related toxicity was observed in 1 of 3 patients, 3 more patients were to be added at the same dose level. Adverse events not judged to be related to CT-011 were not considered as toxicity in terms of these dose escalation and maximum tolerated dose rules.

Subsequent to drug administration, patients were monitored for safety, including adverse events and clinical and laboratory responses at 24 h, 48 h, and on days 7, 14, and 21. When an adverse event occurred, the following information and assessments were recorded: the signs, symptoms, or diagnosis of the event; the date and time of onset of the event; the adverse event severity; the relationship of the event to the study drug; and the description of any action taken with regard to study drug disposition and any required therapy, medication, treatment, or diagnostic procedure. Follow-up of a patient had to be conducted until resolution of the adverse event.

Treatment assessment. The parameters used to assess clinical responses to CT-011 therapy throughout the study were as follows: for leukemia patients, percentage of blasts in the peripheral blood and/or in bone marrow aspirations; for chronic lymphocytic leukemia, size of the lymphatic nodes and the liver, spleen, and Binet scale; for non-Hodgkin's lymphoma and Hodgkin's lymphoma, computed tomography scan to evaluate size and location of masses in lymphatic nodes and other organs; for multiple myeloma, protein electrophoresis, β₂ microglobulin, Immunoelectrophoresis, and Bence Jones protein in the urine.

In addition, recording of survival and general clinical status was conducted for a period of up to 12 mo postenrollment.

Sample collection and pharmacokinetic analysis. Blood samples were withdrawn from all patients before and immediately after CT-011 infusions (in some of the patients) and at 24 h, 48 h (in some of the patients), and on days 7, 14, and 21. CT-011 blood levels were assessed with a specific ELISA assay and derived concentrations were used to generate pharmacokinetics profiles versus time for all patients. All pharmacokinetic analyses were done using SAS for Windows, version 9.1.3, to estimate area under the serum concentration-time curve from time 0 to the final time with a concentration equal to or greater than the limit of quantification [AUC_(0-t)]; area under the serum concentration-time curve from time 0 to infinity [AUC_(inf)]; plasma clearance; maximum plasma concentration (C_max); mean residence time; volume of distribution (V_z); half-life (t_1/2); and time of maximum concentration (T_max). Graphs were generated using SigmaPlot 8.0.

Immune system activation. Activation of the immune system was assessed by measuring blood levels of the immunologic markers CD4, CD8, and CD69 with a fluorescence-activated cell sorter and by measuring the serum levels of tumor necrosis factor α and IFNγ with commercially available kits (Uden and Invitrogen Corporation, respectively).

Statistical analysis. Categorical variables were evaluated by frequencies, percentages, and distribution. Distribution for categorical variables with small numbers of observations (at least one cell with less than five observations) were compared using the Fisher-Irwin exact test. Continuous variables were evaluated by ranges, means, and SDs. The results between the five groups were analyzed by ANOVA. The results between pairs of observations were analyzed with the t test for paired differences. The cumulative incidence estimate was used to calculate the probability of survival rate as a function of time. The log-rank test was used to compare between survival curves.

Data derived from the clinical record forms were recorded and compiled into a database. The database entries were 100% cross-checked with the clinical record form and source documents and transferred to a Stata data file (Statistics/Data Analysis, version 6.0; Stata Corporation). All the statistical processing and analyses were done using Stata software.

Results

The study enrolled a total of 17 patients, whose main characteristics are listed in Table 1 , from May 2004 to April 2005. Screening evaluations were conducted up to 3 days before study drug administration. None of the patients failed the screening evaluation. Patient 003, initially treated at 0.2 mg/kg, requested a repeat compassionate treatment and was treated again at 3 mg/kg. Due to the fact that the interval between the first and second treatment was 5 months, the different treatments were analyzed as separate individuals. Therefore, the number of CT-011 administrations used for the statistical and other analyses was 18. All patients received their assigned dose of CT-011.

Four of the patients received allogeneic stem cell transplantation and one patient received autologous stem cell transplantation 6 to 14 weeks and 19 weeks before CT-011 therapy, respectively. Four patients received irradiation and five others received various chemotherapeutic treatments 1 to 50 weeks and 1 to 99 weeks before CT-011 therapy, respectively (Table 1). One patient received supportive therapy with granulocyte colony-stimulating factor, erythropoietin, and blood transfusions 2 weeks before the first administration of CT-011, and later received a second dose of CT-011 as compassionate treatment. Two patients did not receive any treatment for their disease before being treated with CT-011. The majority of the patients were either asymptomatic or had only mild symptoms at study entry. ECOG performance status, determined at screening and visits 3, 5, 6, and 7, was used to assess how a patient's disease affected the daily living abilities of the patient (Table 1).

Patient enrollment, disposition, and reasons for study withdrawal are summarized by dose group in Table 2 . A cohort of three patients received the initial dose level of 0.2 mg/kg CT-011 by i.v. infusion over 5 hours. Following the administration of the initial dose, CT-011 dose was to be increased by stages to 0.6, 1.5, 3, and 6 mg/kg, or until dose-limiting toxicity was reached. Two patients withdrew from the study following their treatment: one patient in the 0.6 mg/kg group who was unable to attend study visits due to her medical condition and one patient in the 3 mg/kg group who was allocated to stem cell transplantation following the identification of a suitable donor. Four AML patients experienced serious adverse events that resulted in their death.

Tolerability. The primary objective of the study was to determine the safety, dose-limiting toxicity, and maximum tolerated dose of a single i.v. administration of CT-011 to patients with advanced stage hematologic malignancies. All planned dose levels (0.2, 0.6, 1.5, 3, and 6 mg/kg) of the study were administered. No dose-limiting toxicity was reached in the study. CT-011 was found to be safe and well tolerated with no treatment-related toxicities. No single maximum tolerated dose was found in this study.

During the study, 61% (11 of 18) of patients reported adverse events: 67% (2 of 3) of patients in the 0.2 mg/kg dose group, 0% (0 of 3) of patients in the 0.6 mg/kg dose group, 100% (3 of 3) of patients in the 1.5 mg/kg dose group, 67% (4 of 6) of patients in the 3.0 mg/kg dose group, and 67% (2 of 3) of patients in the 6.0 mg/kg dose group. All nonserious adverse events reported during the study are listed in Table 3 .

The most frequent adverse event observed in the study was diarrhea, which occurred in two patients (patients 001 and 014). Patient 014 had presented with diarrhea at the time of enrollment (before any treatment). Patient 001 did receive allograft and it is possible that the diarrhea observed was due to the graft versus host disease that was suspected in this patient already at the time of enrollment (due to rash noted in this patient). Diarrhea was not observed in all other patients that had prior allograft whereas patient 014 received autologous stem cell transplantation. Therefore, we concluded that the observed diarrhea was not associated with CT-011 treatment.

Neither of these adverse events was considered to be related to study drug. Two adverse events, weakness and flushing, were considered possibly related to study drug treatment and occurred in the same chronic lymphocytic leukemia patient. No dose relationship of adverse events could be established.

Four serious adverse events occurred during the study in three female patients and one male patient. All serious adverse events resulted in death of the patient and occurred in AML patients across several study drug doses. Clinical analysis concluded that all of these patients died from fulminate resistant leukemia and none of these deaths was considered to be related to study drug. One patient died from combination of resistant leukemia and grade 4 graft versus host disease This patient, a 64-year-old woman with resistant acute myelogenous leukemia M4, underwent allogeneic stem cell transplantation (stem cell transplantation with granulocyte colony-stimulating factor–mobilized peripheral blood stem cells; i.e., unmanipulated graft which includes both CD34⁺ and CD3⁺) from her HLA-matched donor. Eight weeks after allogeneic stem cell transplantation, the patient was enrolled at the study already presenting with rash suspected as the initiation of graft versus host disease and received the lowest dose planned for this study, 0.2 mg/kg CT-011. Subsequently, the patient developed grade 4 graft versus host disease of the gastrointestinal track associated with diarrhea and, despite intensive supportive and immunosuppressive treatment, she continued to deteriorate.

Analyses of blood chemistry and hematologic parameters on a per dose group as well as across follow-up visits identified only anecdotal changes that did not exhibit a dose or time relationship. No clinically significant changes in any of the vital signs were noted during the 5-hour infusion, during the 4 hours following the end of infusion, or at the follow-up visits. Several scattered changes in various body systems were noted. However, no dose, time, or disease relationship could be determined.

Efficacy and survival. Although this first in-human phase I study was not designed to address questions of efficacy, the following evaluations were done during the study: clinical response, ECOG performance status, and survival. These findings are presented for exploratory purposes only. All patients who participated in the study, with the exclusion of the two patients who withdrew, were evaluated for clinical responses and survival. For the two patients who withdrew, only overall survival data are available. Overall, the ECOG performance status remained stable in 12 patients, increased in 5 patients, and decreased in 1 patient during the 21 days of the study.

Over the 21 days of the study, no change in the average percentage of blasts in the peripheral blood of AML patients was observed with the exclusion of one AML patient that exhibited a reduction in the number of peripheral blasts from 50% to 5%. Additionally, there were no changes in disease parameters during the 21 days of the study in two chronic lymphocytic leukemia patients, four non-Hodgkin's lymphoma patients, and one multiple myeloma patient.

The cumulative survival of all patients (n = 18) at 21 days was 76%, with a 95% confidence interval of 48% to 90%. No difference in mean survival time across the dose groups was noted.

Patients were followed for survival beyond the 21 days of the study. The mean survival time in the study was 25 ± 27 weeks, ranging from 1.7 to >77 weeks. This follow-up suggested that six patients exhibited apparent response to treatment with extended survival averaging at least 60 weeks (three patients are still alive). These patients are represented in Table 4 . There was one complete remission in patient 015 that received the fourth dose level of 3.0 mg/kg. This patient was diagnosed with stage III follicular lymphoma involving nodes below and above the diaphragm. The patient did not receive any prior treatment for her disease. In a computed tomography scan done during a periodic check 10 months after CT-011 treatment, complete elimination of tumor masses was observed. Interestingly, the patient did not receive any further treatment during the period that lapsed between CT-011 treatment and the 10-month check. The patient has shown a sustained remission 68 weeks following CT-011 treatment. One minimal response was observed in an AML patient receiving CT-011 at 0.2 and 3 mg/kg. The patient progressed 61 weeks after receiving CT-011. Four patients have shown stable disease: One with Hodgkin's lymphoma receiving CT-011 at 0.6 mg/kg had a stable disease for 35 weeks. Two patients with chronic lymphocytic leukemia receiving the antibody at 0.6 and at 1.5 mg/kg were stable for 36 and >78 weeks, respectively. A multiple myeloma patient receiving CT-011 at 6.0 mg/kg showed stable disease for >60 weeks.

Pharmacokinetic evaluation. A secondary end point of the study was to determine the pharmacokinetics of CT-011. Blood samples for pharmacokinetic analysis were to be drawn on day 0, at 24 and 48 hours postinfusion, and on days 7, 14, and 21. Serum was harvested from the blood and CT-011 concentrations were determined by a specific ELISA method. Figure 1A depicts the mean plasma concentrations of the antibody with time following the administration of different doses of the antibody, showing an initial fast distribution phase that is followed by a gradual elimination phase. Mean values for C_max, AUC_(0-t), and AUC_(inf) increased in an apparent dose-related manner. As illustrated in Fig. 1B, log-log plots of C_max and AUC_(inf) were linear with slopes approximately equal to 1, indicating linearity of CT-011 pharmacokinetics. The analysis further indicated that the mean clearance ranged from 0.14 to 0.34 mL/h/kg and the mean volume of distribution (V_z) ranged from 53.1 to 90.3 mL/kg. Within the limitation of the small number of patients per dose, there were no apparent differences among doses in the clearance of CT-011 (see Fig. 1C). In addition, there were no apparent differences between male and female patients or among patients with AML, myelodysplastic syndrome, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, Hodgkin's lymphoma, or multiple myeloma in the pharmacokinetics of CT-011 (data not shown).

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Fig. 1.

CT-011 pharmacokinetics. A, relationships between mean C_max, AUC _(inf), and dose as a function of dose administered. B, relationships between mean C_max, AUC_(inf), and dose after i.v. administration of CT-011 to patients with hematologic malignancies. C, individual patient serum clearance (CL) by dose after i.v. administration of CT-011 to patients with hematologic malignancies.

The median serum t_1/2 of CT-011 ranged from 217 to 410 hours (9-17 days) and is within the expected ranges found for other mAbs in humans. The median t_1/2 for the six patients with an apparent clinical benefit to treatment seems to be higher than that of the rest of the patients [353 versus 200 hours (14.7 versus 8.4 days), respectively]. These differences did not reach significance, most probably due to the small number of patients.

Immune system activation. Activation of the immune system was assessed by measuring levels of the immunologic markers CD4, CD8, and CD69 and by measuring the serum levels of tumor necrosis factor α and IFNγ. Blood samples were to be drawn at screening, 24 hours postinfusion, and on days 7, 14, and 21.

Table 5 summarizes the median values of percentage of peripheral blood CD4⁺, CD8⁺, and CD69⁺ lymphocytes at the different time points they were evaluated during the trial. Elevations in the percentage of peripheral blood CD4⁺ lymphocytes were observed already at 24 hours after CT-011 treatment in 15 of the patients. The overall magnitude of the elevation at the 24-hour time point was 37% (median), sustaining a range of 11% to 30% till day 21. This increase was found to be statistically significant in patients treated with 0.6 mg/kg (increased from mean level of 22% to 34%; P < 0.02) and 3 mg/kg (increase from mean level of 33% to 42%; P < 0.005) of CT-011. The percentage of peripheral blood CD8⁺ lymphocytes fluctuated around the initial level at a magnitude of 6% up to the last sampling at day 21. A statistically significant decrease was noted in patients that were treated with 3 mg/kg CT-011 (decreased from mean level of 32% to 27%; P < 0.05). No significant changes in peripheral CD69⁺ lymphocytes were observed.

No change in the levels of IFNγ or tumor necrosis factor α was noted in sera derived from hematologic malignancy patients following treatment with CT-011 at any of the time points (data not shown).

Discussion

One of the leading causes for immune suppression in cancer patients was suggested to be associated with the elevated expression of PD-L1 (B7-H1) at tumor-involved sites, resulting in local suppression and apoptosis of tumor-infiltrating effector lymphocytes. Recent clinical data support notion of PD-L1 (B7-H1) playing an important role in immune evasion of tumor cells (9). Thus, we hypothesized that targeting B7-H1/PD-1 interactions and, specifically, interference with the function of PD-1 by mAbs may potentially be useful in enhancing immunity against cancer antigens.

Here, we report the results of a phase I clinical study with CT-011 in 17 patients with advanced stage hematologic malignancies. CT-011 is a humanized IgG1 mAb that modulates the immune response through its interaction with the PD-1 receptor. The study showed the antibody to be safe and well tolerated in this patient population and no single maximum tolerated dose was determined.

The cumulative survival of all patients at 21 days was 76%, and follow-up beyond the 21 days of the study revealed a mean survival of 25 weeks. Given that most of the patients were at an advanced stage of their disease, it was encouraging to find that six patients exhibited apparent response to treatment with extended survival averaging 60 weeks.

Pharmacokinetic analyses have shown that serum C_max and the AUC of CT-011 increased proportionally with dose. There were no apparent differences between male and female patients or among patients with AML, myelodysplastic syndrome, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, Hodgkin's lymphoma, or multiple myeloma diagnosis in the pharmacokinetics of CT-011. The median t_1/2 of CT-011 ranged from 217 to 410 hours (9-17 days), which is within the expected range found for other mAbs in humans. Interestingly, the median t_1/2 for the six patients with apparent clinical response to treatment was somewhat higher than that of the rest of the patients. The association of clinical efficacy with extended half life has also been shown for other antibodies (e.g., CP-675,206, an anti–CTLA-4 antibody; Rituximab) but is not yet understood and thus should be evaluated for possible predictive biomarkers in future clinical studies. Taking into account the duration of the response with an average of 60 weeks in these six patients and the pharmacokinetics of the antibody with a highest half life of 410 hours, we assume that in some of the patients, tumor-specific immunologic memory has been induced leading to durable antitumor immune response long after the antibody has been eliminated from the blood.

It was recognized that B7-H1 is constitutively expressed by human cancer tissues (10–13). Several lines of evidence indicate that B7-H1 is involved in tumor-induced immune evasion (14). One study showed that tumor-associated B7-H1 could prevent antitumor immune response by inducing apoptosis of effector T cells in vivo (10). B7-H1 expressed on tumors was shown to interact with the coinhibitory receptor PD-1 that functions in effector/memory T lymphocytes and plays a regulatory role in peripheral tolerance (1, 15). Therefore, we hypothesized that treatment with CT-011 may interfere with this mechanism and thereby affect the level of peripheral blood CD4⁺, CD8⁺, and CD69⁺ lymphocytes. The observed elevations in the percentage of peripheral blood CD4⁺ lymphocytes noted already at 24 hours after CT-011 treatment may be related to inhibition of apoptosis of effector T cells by the drug. In addition, this observation may also be attributed to homing of T cells to tumor-involved sites. CD69 is the one of earliest activation markers newly synthesized and expressed during T lymphocyte activation. We were not able to find significant changes in CD69⁺ lymphocytes related to CT-011 treatment.

In summary, a single administration of 0.2 to 6.0 mg/kg of CT-011 is safe and well tolerated in patients with advanced hematologic malignancies. The observed clinical benefits may be related to durable tumor-specific immune response that is induced by CT-011. Based on data obtained from this trial, elevations in the percentage of peripheral blood CD4⁺ lymphocytes may serve as a potential biological marker for future trials. A phase II clinical study evaluating the safety and efficacy of CT-011 administered at the dose level of 1.5 mg/kg in diffuse large B-cell lymphoma following autologous bone marrow transplantation has been initiated. CT-011 dose was chosen based on the pharmacokinetic analysis done in this phase I study as well as on comparability to efficacy and pharmacokinetic parameters obtained in studies conducted in animal models.

Disclosure of Potential Conflicts of Interest

R. Rotem-Yehudar, G. Slama, and S.Lendes are employees of Curetech, Ltd.