Clinical safety and pharmacological profile of the HLA-DR antibody 1D09C3 in patients with B cell chronic lymphocytic leukemia and lymphoma: results from a phase I study

Abstract

1D09C3 is a human monoclonal IgG4-type antibody against human leukocyte antigen-DR (HLA-DR) which has demonstrated pro-apoptotic activity against lymphoid tumors in vitro and in vivo. We report results from a phase I dose-escalation study which aimed to identify tolerated dosing, and the pharmacokinetic and pharmacodynamic profile of 1D09C3. Fourteen patients with relapsed/refractory B cell type leukemia/lymphoma were treated and followed after up to 4 weekly infusions of 1D09C3, administered in 6 dose levels at 0.25–8 mg/kg/day. Treatment was tolerated well with mostly mild side effects. The most common grade III–IV toxicities were hematological events observed in 4 patients. In one patient, treated at 8.0 mg/kg/day, a dose limiting toxicity occurred, identified as an invasive catheter-related infection. Adverse events resolved completely without long-term sequelae. 1D09C3 reduced peripheral blood B cells and monocytes by a median of 73–81 % in all patients, with a nadir reached 30–60 min after infusion and sustained for <96 h. Granulocytes and natural killer cells predominantly increased with variable time courses. Pharmacokinetic assessments showed detectable drug concentrations at doses 4–8 mg/kg/day and a terminal half-life of 0.7–7.9 h. Effective saturation of HLA-DR on peripheral blood B cells/monocytes was achieved, varying consistently with available serum concentrations and the cell-reducing activity of 1D09C3. In summary, 1D09C3 could be administered safely in patients with advanced B cell malignancies. Pharmacodynamic studies demonstrated a strong dose dependent but transient reduction of peripheral blood B cells and monocytes, consistent with a short drug serum availability.

Electronic supplementary material

The online version of this article (doi:10.1007/s00262-012-1362-x) contains supplementary material, which is available to authorized users.

Introduction

In vitro and in vivo crosslinking of human leukocyte antigen-DR (HLA-DR) by murine antibodies has been found to induce significant growth inhibition or apoptosis in activated normal and neoplastic B cells [1–3]. 1D09C3 is a fully human monoclonal antibody, specifically developed from the Human Combinatorial Antibody Library (HuCAL) as an IgG4-subtype immunoglobulin to reduce effector functions and potential side effects mediated by the Fc portion [4, 5]. In contrast to other HLA-DR antibodies (i.e., L243, 8D1), 1D09C3 was shown to exert a direct pro-apoptotic effect on target cells, which was confirmed to be independent of complement- or effector cell-mediated cytotoxicity (CDC/ADCC) in vitro [5].

1D09C3 showed promising anti-tumor activity in various murine xenotransplant models, that is, for human Hodgkin’s and Non-Hodgkin’s lymphoma, hairy cell leukemia, myeloma and B cell pro-lymphocytic leukemia [5–7]. Preclinical toxicity studies in Cynomolgus monkeys demonstrated rapid clearance of the antibody from peripheral blood with a terminal half-life of 35–140 h [8]. At high-dose administrations, serum levels of 1D09C3 were traceable in the animals for up to a week, which recommended a weekly dosing schedule for first clinical testing in humans. Concomitant histopathologic studies in primates revealed a cumulative depletion of B lymphocytes from lymph nodes and spleen associated with low levels of T cell infiltration, while no severe changes were detected in other tissues [5, 6, 8].

Here, we report data from a first open-label phase I dose-escalation study in man which was designed to determine 1. the maximum tolerated dose (MTD) and dose limiting toxicity (DLT), and 2. to characterize the pharmacokinetic and pharmacodynamic profile of 1D09C3 in humans.

Design and methods

Patient eligibility

Fourteen patients with CLL, Hodgkin or Non-Hodgkin’s lymphoma (HL/NHL) were enrolled on a single-center phase I study of 1D09C3 at the University of Cologne, Germany (EudraCT-No 2005-004931-23), after informed consent had been obtained. The trial was approved by the local ethics committee of the Medical Association Nordrhein in Düsseldorf and by the Federal Institute for Vaccines and Biomedicines (Langen, Germany). All patients presented with relapsed and/or refractory disease and diagnoses confirmed according to National Cancer Institute (NCI) working group and/or World Health Organization criteria [9, 10]. A complete list of inclusion/exclusion criteria is provided as supplementary material.

Trial design, treatment and follow-up

The study treatment included 4 two-hour infusions of 1D09C3 dissolved in 100 or 250 ml 0.9 % saline, administered on day 1, 8, 15 and 22. Patients were assigned to one dose level, according to a “3 + 3” scheme with doses escalated from 0.5 mg/kg/day. One patient (C101) was treated at 0.25 mg/kg/day, an additional safety dose level introduced below the recommended start dose of 0.5 mg/kg/day established from animal studies. Vital signs, adverse events and laboratory parameters were monitored throughout the infusions and at various time points on day 1, 2, 3, 4 and 6 of each treatment week and on days 29, 36, 50, 57 and 64. Radiographic studies to assess lymphadenopathy were performed prior 1D09C3 and on days 29 and/or 50 (+/− 7 days). Three-monthly follow-up visits were carried out for up to 1 year. The overall tumor response was evaluated according to NCI criteria [9, 11].

Trial endpoints and safety criteria

Primary endpoint of the study was the determination of the MTD and DLT for 1D09C3. The following criteria were used to define a DLT: 1. inability to administer consecutive doses of 1D09C3 on day 1, 8, 15 and 22 due to any toxicity; 2. occurrence of disseminated intravascular coagulation ≥grade 3; 3. signs of coagulation toxicity defined as fibrinogen <50 % of LLN, INR/PTT >200 % of ULN; 4. any non-hematological toxicity ≥grade 3 with the exception of vomiting in the absence of appropriate antiemetic therapy, hypersensitivity reactions and diarrhea in the absence of optimal prophylaxis/antidiarrhetic drugs. The MTD was defined as the last dose level below the dose, where at least 2 of 6 treated patients develop a DLT.

Laboratory testing

Absolute and relative counts of white blood cell subsets were investigated by flow cytometry using standard fluorochrome-labeled antibodies and TruCOUNT tubes (Beckman-Coulter, Krefeld; Becton Dickinson, Heidelberg, Germany). 1D09C3 serum concentrations were measured with a validated enzyme-linked immunosorbent assay and evaluated based on a non-compartmental model for intravascular constant infusion (Biopharm, Eppelheim; BioProof, Munich, Germany). Saturation of HLA-DR epitopes by 1D09C3 was measured on B cells and monocytes by flow cytometry using indirect staining of HLA-DR-bound 1D09C3 with a fluorochrome-conjugated anti-human-IgG4 antibody (Huntingdon Life Sciences, Huntingdon, Cambridgeshire, UK). Saturation levels at a given time point were calculated relative to the maximum HLA-DR binding of 1D09C3, measured indirectly as anti-IgG4 binding on target cells after ex vivo oversaturation with 10 μg/mL 1D09C3.

Data collection and analyses

Clinical and laboratory data were collected in accordance with German privacy protection laws in case report forms at the University Hospital of Cologne. Standard statistical tools were used to compare clinical/laboratory features in different patient groups using Stata (College Station, Texas, USA). Pharmacokinetic evaluations were computed in WinNonlin (Pharsight Corporation, St. Louis, MO, USA).

Results and discussion

Fourteen patients (10 CLL, 3 NHL, 1 HL) at a median age of 67 years and with median 5 pre-treatments were enrolled and dosed with 1D09C3 (0.25–8 mg/kg/day) as outlined in Table 1. Overall, 1D09C3 therapy was administered safely and tolerated well. CTC grade 3–4 events were mostly hematological side effects observed in four patients (dose 1.0–8.0 mg/kg/day). Ten events of thrombocytopenia, 3 anemias and 3 neutropenias occurred within 24 h after 1D09C3 infusions with a maximum duration of 4 days. They resolved unassistedly (13 events) or were successfully treated with transfusions (3 events of anemia). Importantly, all 4 patients had corresponding disease-related CTC grade 1–2 cytopenias pre-existing prior to the start of 1D09C3 therapy. While activated neutrophils might express HLA-DR [12], the adverse effect of 1D09C3 on red blood cells and platelets in these cases cannot be explained by HLA-DR crosslinking.

Table 1.

Patient characteristics, 1D09C3 dosing and outcome

Patient code	Sex	Age	Diagnosis	Years from 1st diagnosis	Stage	Post-treatment status	Number of pre-treatments	Time between end of previous treatment and start 1D09C3 (months)	1D09C3 dose (mg/kg/ day)	Number of 1D09C3 infusions	Response day 29 (1 week after last 1D09C3)	Response day 50 (1 month after last 1D09C3)	Time to next regimen after last 1D09C3 (weeks)	Overall survival statush	Time of death or last follow-up after start 1D09C3 (months)
C101	f	57	CLL	15	C/IV	Refractory	8	2	0.25	4	SD	SD	10	Deceased	11
C102	m	67	CLL	10	C/IV	Refractory	4	2	0.5	4	–	SD	9	Deceased	34
C103	m	62	CLL	5	C/IV	Relapsed	2	48	0.5	4	PD	–	7	Deceased	29
C104	m	69	CLL	14	C/IV	Refractory	5	8	0.5	4	SD	SD	7	Deceased	18
C105	m	69	CLL	9	C/III	Relapsed	3	36	1	4	SD	PD	6	Deceased	18
C106	m	64	NHLa	17	III	Relapsed	6	24	1	4	PD	–	Noneg	Deceased	9
C107	m	75	NHLb	9	IV O	Refractory	5	0c	1	4	SDf	PD	Noneg	Deceased	2
C108	m	66	CLL	2	B/II	Refractory	5	2	2	3d	SD	–	Noneg	Deceased	3
C109	f	64	CLL	5	C/IV	Refractory	5	8	2	4	–	PD	15	Deceased	8
C110	m	64	CLL	8	B/II	Relapsed	3	36	2	4	PD	–	10	Deceased	43
C111	m	45	NHLa	5	IV	Relapsed	5	16	4	2d	–	SD	–	Alive	55
C112	f	74	CLL	12	C/IV	Refractory	8	5	4	4	–	PD	Noneg	Deceased	3
C113	m	73	CLL	1	C/IV	Refractory	1	6	4	4	SD	PD	7	Deceased	8
C114	m	71	HL	3	II B	Refractory	3	5	8	1e	SD	SD	9	Deceased	21

The disease stage is given either according to the Binet and Rai staging system for CLL patients, or according to the Ann-Arbor staging system for lymphoma patients. The median time from diagnosis to enrollment was 8.5 years (1–17 years). At final data analysis and a median follow-up of 14.6 months (range, 2.0–54.9), calculated from the start of 1D09C3, 13 patients had died of disease progression

Information for empty cells with “–” was not available/obtained

ECOG status Eastern Cooperative Oncology Group performance status, f female, m male, PD progressive disease, SD stable disease

^aFollicular Non-Hodgkin’s lymphoma

^bDiffuse large B cell Non-Hodgkin’s lymphoma

^cPatient received repeated courses of trofosfamide and steroids within 4 months up to 1 week prior to start of 1D09C3 to reduce symptomatic lymph edema in his right leg

^dPremature stop due to patient’s decision

^ePremature stop due to dose limiting toxicity

^fStaging was performed on day 35

^gPatient went on supportive care

^hThe overall survival status was obtained when the manuscript was written

In one patient (C114), 1 day after the first 1D09C3 infusion (8 mg/kg/day) fever of unknown origin occurred and was ruled as a DLT with subsequent stop of 1D09C3 treatment. Further diagnostics, however, revealed recurrent bacteremias due to an infected central catheter (port), retrospectively unrelated to study treatment. Unfortunately, the trial was stopped prematurely after treatment of patient C114 due to the manufacturer’s decision to not further pursue 1D09C3 as a product. Therefore, an MTD for 1D09C3 could not be determined.

Clinically, 1D09C3 demonstrated limited activity: On day 29 and/or at day 50 (1 week or 1 month after last 1D09C3 infusion (Table 1), 6 patients presented with stable disease and 8 patients with disease progression. In contrast, peripheral blood immunophenotyping by flow cytometry demonstrated interesting activity of the study drug, consistent with pre-clinical data: a strong but short-lasting decrease of peripheral white blood cells after 1D09C3 therapy was noted with a characteristic infusion-related time course observed in all 14 patients (Fig. 1a). The median leukocyte nadir was reached in most cases 30–60 min after termination of each 1D09C3 infusion, however, counts returned rapidly within 24 h with some excess rebound detectable for 2–8 days.

Fig. 1.

Time course of peripheral white blood cell subsets (a and b) and HLA-DR saturation (c and d) after 1D09C3 therapy, as determined by flow cytometry. The x-axis counts the days from start of 1D09C3 treatment with the first 1D09C3 infusion administered on day 1. The second, third and fourth 1D09C3 infusion were given on days 8, 15 and 22. In figures a and b, the y-axis represents the percent fractional change of each cell population calculated relatively to the baseline level (=100 %), which was measured before the first 1D09C3 infusion was administered. Each cell population was determined by a characteristic surface marker profile. In figures c and d, the y-axis represents the relative mean fluorescence intensity of 1D09C3 bound to CD19+ B cells (c) or CD14+ monocytes (d). Here, the level of 100 % marks the maximum possible binding of 1D09C3, achieved by experimental oversaturation of all HLA-DR epitopes with 1D09C3

Leukocyte subtyping (Fig. 1b) demonstrated that most cell subsets, except granulocytes, followed this particular time course with the highest number of cell loss occurring in CD19-B cells (median 81.6 %, range 34.8–98.9 %) and monocytes (median 73.0 %, range 15.0–94.7). Granulocyte counts showed no clearly infusion-related pattern with predominantly increasing levels (overall median peak 206.7 %, range 107.5–882.2 %), reached at variable time points. NK cells showed a heterogeneous but yet infusion-related time course with a median reduction by 24.0 % (range, 80.2 to +124.6 %) and excessive rebounds up to 103.3–1,250.3 % relative to the baseline value (overall median peak 253.3 %, reached at variable time points). The relative loss of B cells after 1D09C3 infusion was higher than depletion of any other cell subset analyzed and with the slowest recovery to baseline levels (P ≤ 0.0071, pairwise Wilcoxon test, Fig. 1b). There was no difference in the relative B cell decrease in CLL versus lymphoma patients without malignant B cell lymphocytosis.

Besides the consideration of potential underdosing of 1D09C3 in our study, the rapid reappearance of ostensibly depleted blood cells in patients also raised concerns about the bioavailability of the antibody, HLA-DR binding and the possibility of other intravascular effects rather than genuine killing of cells. Pharmacokinetic analyses in our trial demonstrated sustained serum appearance of 1D09C3 at doses ≥4 mg/kg/day, dose-dependent peak concentrations at 2.0–2.3 h and rapid clearance from peripheral blood (terminal half-life 0.7–7.9 h).

In ex vivo analyses of 1D09C3 binding to HLA-DR in 10 treated CLL patients, we found an infusion-dependent time course with dose-dependent surface binding of 1D09C3 to CLL cells and monocytes in all patients. Peak saturations were reached at 30 min after the end of infusion, followed by a rapid decrease after 1 h and a return to baseline levels on day 2–4 after treatment (Fig. 1c/d). Further, increased saturation levels of HLA-DR epitopes on CLL cells/monocytes correlated with higher drug serum levels and with achieved relative cell depletion in these subsets (supplementary files, available online). Thus, the cell-reducing activity of 1D09C3 seemed to require presence of a distinct drug serum concentration and HLA-DR binding level; however, it was probably terminated by a limited serum availability and/or activity of 1D09C3. We did not find an association between 1D09C3 pharmacokinetics and peripheral blood tumor load (WBC/CLL B cell count) or immunogenicity of the drug (human anti-human antibodies, supplementary files) in patients treated with the same 1D09C3 dose.

Protein therapeutics in general tend to have an extended half-life in peripheral blood, for example, 14–62 days for rituximab in CLL patients [13]. Considerable biological processes possibly causing inactivity and/or instability of antibodies are, that is, shedding of the target antigen from the cell surface with complex formation in the blood stream, (non-)enzymatic modification/degradation (i.e., deamidation, oxidation), margination/aggregation or internalization. IgG4-type antibodies have been reported to be prone to form half-antibodies undergoing exchange of Fab fragments with endogenous immunoglobulins in vivo [14, 15]. Due to an increasing number of reports on this phenomenon, the manufacturer decided to halt clinical development of 1D09C3. Hansen and coworkers recently demonstrated that 1D09C3 is indeed able to undergo Fab-arm exchange with other IgG4 molecules in a distinct redox environment in vitro [16]. Thus, this effect, caused by a rearrangement of disulfide bonds in the hinge region, might have compromised the activity of 1D09C3 in our study.

Hansen and colleagues also showed that co-incubation with the murine HLA-DR antibody L243 or 1D09C3 leads to the aggregation of B cell lymphoma cell lines in vitro. The authors claimed that rather the mechanical disruption of such aggregates for further experimental processing, that is, for flow cytometry, rather than induced programmed cell death, subsequently kills the cells. These data question the actual mechanisms and cellular consequences of 1D09C3- and HLA-DR-induced signaling, as established by various groups working with 1D09C3 or other HLA-DR antibodies in vitro or in vivo [1–3, 5, 6, 17, 18]. However, a potential overrating of the cell-killing activity of HLA-DR antibodies observed in vitro—as suggested by the data from Hansen and colleagues—does not explain the promising therapeutic effects of these drugs achieved in animal tumor models.

Ivanov et al. [19] have found that a homotypic adhesion of cells is an important pre-requisite for subsequent cell death, induced by crosslinking antibodies such as the CD20 antibody tositumomab or L243. Using electron microscopy-based studies, they found a reorganization of actin filaments, membrane rafts and finally lysosome-dependent disintegration of plasma membranes following to tositumomab or L243 treatment of CLL cells or cell lines [19]. Thus, albeit these experiments were not completed without some kind of mechanical manipulation of cells, HLA-DR crosslinking seems to be able to induce cellular changes potentially leading to spontaneous cell death after the formation of cell aggregates. Possibly, 1D09C3 induced such intravascular aggregates or adhesion of cells to endothelium, also involving platelets and/or red blood cells (possible reasons for anemia/thrombocytopenia). However, the exact mechanisms and consequences of intravascular and cellular HLA-DR signaling seem to be still poorly understood to draw final conclusions.

To date, several HLA-DR antibodies have been developed, two of which have been studied in a phase I/II setting. None of these studies provided a detailed characterization of drug activity on human blood leukocyte subsets, as presented in this report. Further, the functional activity of these antibodies relied at least in part on intact patient effector mechanisms like CDC/ADCC. A stepped-up dose-escalation trial investigating the safety and tolerability of apolizumab (Hu1D10, Remitogen), a humanized murine IgG1 monoclonal antibody directed against the HLA-DR β-chain, has recently been reported by Lin et al [20]. The MTD of Hu1D10 in 23 patients (22 CLL, 1 ALL) was defined at 3.0 mg/kg/day, similar to previous experience with this antibody collected in follicular lymphoma [21]. The antibody demonstrated a fairly long half-life of 8–36 days in patients. However, the overall clinical response was not convincing and the antibody discontinued by the manufacturer. A second drug, the murine IgG2a-type antibody Lym-1 which also targets the HLA-DR β-chain, was tested in a phase I study including 10 patients with refractory B cell lymphomas with reasonable tolerability. However, penetration of the drug into extravascular tissue was low and clinical responses minor [22].

A third humanized IgG4-type anti-HLA-DR antibody, IMMU-114, has been under pre-clinical testing by Stein and coworkers [17, 18]. The investigators recently reported a pre-clinical canine model, where IMMU-14 demonstrated a rapid clearance from peripheral blood within hours after infusion, similar to 1D09C3 [23]. In contrast to 1D09C3, the antibody contains a mutated hinge region in order to prevent the formation of half-antibodies. Thus, mechanisms other than Fab exchange must affect the bioavailability of this study drug which has not entered clinical testing yet.

Overall, our phase I clinical experience in patients with B cell malignancies demonstrated a safe tolerability but transient activity and bioavailability of 1D09C3 in humans at dose levels tested in this trial. The observed short-term reduction of predominantly B cells and monocytes in peripheral blood needs to be interpreted in context of a limited dosing schedule, potential loss of activity of 1D09C3 due to IgG4-specific structural features (Fab-arm exchange) and with consideration of margination or aggregation as consequences of HLA-DR crosslinking. Clinically, sole HLA-DR-specific cancer therapy seems to be not convincingly successful with monoclonal antibodies and dosing schedules available so far. Bi- or poly-specific target strategies, such as the antibody-coupled immunocytokine 20-C2-2b (combines Fab fragments against HLA-DR and CD20 with interferon-α as a toxic cytokine) [24] or single-chain triplebodies [25], are currently investigated to exploit the high in vivo binding efficiency achieved by HLA-DR antibodies, as it was evident for 1D09C3 from our study. However, our and published data raise important questions about the true cellular effects of HLA-DR-derived signals in vitro and in vivo which warrant further experimental clarification.

Electronic supplementary material

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