AFM11-101 was a phase 1 open-label, first-in-human, dose-escalation study conducted at 10 sites in Germany, Czech Republic, Poland and the United States between October 2014 and September 2018. The primary objective of the study was to determine the safety and tolerability of AFM11 administered intravenously at weekly intervals over a period of 4 weeks (1 cycle) in patients with NHL, investigating escalating doses and different infusion durations. Secondary objectives included determination of the maximum tolerated dose (MTD; highest dose at which <33% of patients experienced a dose-limiting toxicity [DLT; any Grade 3 or higher non-haematological toxicity or Grade 4 or higher haematological toxicity or any treatment delay ≥21 days because of drug-related adverse events (AEs)]), with the overall aim of identifying the dose and infusion time for a phase 2 study.
This study included male or female patients ≥18 years with indolent or aggressive CD19+ NHL, who had relapsed or were refractory to standard therapy, which must have included treatment with rituximab plus chemotherapy, and were not eligible for a curative treatment option. All patients must have had measurable disease (at least one lesion ≥1.5 cm) documented by a computed tomography (CT) scan, an Eastern Cooperative Oncology Group (ECOG) performance status of ≤2, and a life expectancy of ≥6 months.
Patients were excluded if their total number of B cells (healthy and malignant combined) in peripheral blood exceeded the upper limit of normal in healthy individuals (assessed by flow cytometry) or if they had central nervous system (CNS) involvement, a history of malignancy other than B-cell lymphoma within 5 years before study entry, active autoimmune disease requiring immunosuppression or uncontrolled infection. Patients who received an autologous haematopoietic stem cell transplant or treatment with alemtuzumab within 12 weeks of the start of AFM11 treatment, chemotherapy, radiotherapy, therapy with an antibody, an investigational drug or corticosteroid treatment within 4 weeks of the start of AFM11 treatment, or who had prior treatment with a CD19-targeting T-cell engager, including CD19 CAR-T cells, were also excluded.
The study used an open-label, dose-escalating design, with the starting dose selected based on the minimum anticipated biological effect level determined from in vitro data and PK models as no relevant in vivo data were available. AFM11 was planned to be escalated at the predefined doses of: 0.0003, 0.001, 0.003, 0.01, 0.03, 0.09, 0.20, 0.40, 0.80, 1.5, and 2.5 µg/kg. Escalation to the next dose cohort only occurred when patients receiving the prior dose level had completed Cycle 1. Patients received the prior cohort dose in Cycle 1 Week 1 and escalated to their cohort dose in Cycle 1 Weeks 2–4. Intrapatient dose reduction was only permitted if a patient experienced cytokine release syndrome or neurotoxicity; intrapatient dose escalation only occurred between Cycle 1 Week 1 and Cycle 1 Week 2, and dose escalation beyond the assigned cohort dose was not permitted. Dosing could be delayed by up to 21 days in each cycle to allow toxicity to return to Grade 1 or baseline. If a dose could not be given within 21 days of the scheduled date, the patient was withdrawn from the study.
Initially, a dosing frequency of 5 times/week in Week 1 and 3 times/week in Weeks 2–4 was selected based on animal models that demonstrated more intensive dosing produced the most effective inhibition of tumour growth. After the first five patients were treated, dosing frequency was reduced to once weekly as this was expected to result in a more favourable safety profile.
AFM11 was given as an intravenous infusion over 4, 24 or 48 hours in a stepwise manner with a separate dose escalation scheme for each infusion time until an MTD had been reached. For the initial 4-hour infusion step, an accelerated titration design (12) with 1 patient per cohort was used until a DLT or activity (B-cell depletion or an increase in interleukin-2 [IL-2], IL-6 or interferon-γ [IFNγ]) was observed, at which point a standard escalation method with 3–6 patients per cohort was used. If a DLT occurred during the accelerated titration phase at dose levels of 0.0003, 0.001, 0.003 or 0.01 µg/kg, the trial immediately transitioned to the next infusion time.
Patients were hospitalised for at least 24 hours or the duration of the infusion. At PK sampling visits, patients were hospitalised for 24, 48 or 72 hours for the 4-, 24- and 48-hour infusions, respectively. Patients who were clinically stable or showed either B-cell depletion or T-cell expansion after Cycle 1, and who had no unacceptable toxicity could receive a second cycle of therapy at the investigator’s discretion. Based on either investigator decision or patient request, patients could be withdrawn from the study at any time.
After the first 5 patients had been treated, the protocol was amended to include mandatory premedication with dexamethasone 20 mg (or equivalent) and antihistamines 1 hour prior to the first and second infusions of AFM11 in Cycle 1 and with dexamethasone 10 mg (or equivalent) and antihistamines 1 hour before all subsequent infusions. Any other medication necessary for the patient’s wellbeing was given at the discretion of the investigator.
AEs were graded according to Common Terminology Criteria for Adverse Events (CTCAE) version 4.03, with assessments including clinical examinations, the assessment of AEs (especially DLT), and laboratory parameters. AEs were captured from the time of consent until 4 ± 1 weeks from the end of the last infusion of AFM11. The investigator assessed whether the AE was not/unlikely related, possibly related, probably related, or definitely related to AFM11. Serious AEs (SAEs) were defined as an untoward medical occurrence that resulted in death, persistent or significant disability/incapacity, was life-threatening, required hospitalisation, was a congenital anomaly or birth defect, or an important medical event as decided by medical and scientific judgement.
Tumour imaging by fluorodeoxyglucose-positron emission tomography and CT was conducted at screening and at 2 (±1) weeks after the end of Cycle 1. Responses were determined by the investigator in accordance with Cheson criteria (13). In patients with bone marrow involvement at baseline, a bone marrow biopsy was required to confirm a complete response.
Blood samples for determination of AFM11 serum concentration were drawn pre-dose and at regular intervals after the start of the infusion until 72, 72 and 48 hours after the end of the 4-, 24- and 48-hour infusions, respectively, in Weeks 1 and 4 of Cycle 1.
Pharmacodynamic analysis included a modified standard panel for lymphocyte analysis by flow cytometry to characterise the basic lymphocyte subset (absolute and relative CD3+, CD4+, CD8+ T cells and the CD4:CD8 ratio, as well as CD20+ B cells). Inclusion of absolute and relative CD16+/CD56+ and CD3- and CD3+ natural killer (NK) cells was optional. In Germany, and optionally in other centres, the expression of activation and memory markers on T cell subsets was assessed by flow cytometry.
Other pharmacodynamic assays included systemic cytokine release, assessed by multiplex analysis of a panel of proinflammatory cytokines, at pre-dose, during infusion and post-dose, quantification of the cytolytic potential of T cells pre- and post-dose, and exploratory biomarker analysis pre- and post-dose with an additional sample to be taken in case of a ≥Grade 3 neurological event.
Antidrug antibodies (ADAs) were measured by an enzyme-linked immunosorbent assay before the first dose and at the Tumour Assessment Visit.
The safety population included all patients who had received at least one dose of AFM11. The PK population was defined as all patients who received at least one dose of AFM11 and for whom serum concentrations of AFM11 were measured.
AFM11-102 was a phase 1, multicentre, open-label dose escalation study of AFM11 in patients with R/R CD19+ adult B-precursor ALL conducted at 12 sites in five countries (Austria, Czech Republic, Israel, Poland, and Russia) between October 2016 and May 2019. The primary objectives were to determine the MTD and to evaluate the safety and tolerability of increasing doses and different infusion times of AFM11 in patients with ALL. Secondary objectives included assessment of the PK and antitumour activity of AFM11.
The study included men or women aged ≥18 years with a diagnosis of R/R CD19+ B-precursor Philadelphia-chromosome negative ALL who were not candidates for bone marrow transplantation with curative intent. CD19 expression must have been confirmed by either staining or flow cytometry of a recent bone marrow biopsy. Patients were required to have failed or be intolerant to therapy with at least two tyrosine kinase inhibitors, have >5% blasts in their bone marrow, an ECOG performance status of ≤2, and a life expectancy of ≥3 months.
Patients were excluded from the study if they had received autologous or allogeneic haematopoietic stem cell transplant within the previous 3 months, had active graft versus host disease, had received prior treatment with blinatumomab or other CD19 targeting T-cell engager, had been treated with a donor lymphocyte infusion, cancer chemotherapy, an antibody or antibody construct, any investigational agent or had received regular corticosteroids or other immunosuppressive in the 4 weeks prior to study entry. Patients with CNS involvement, a history of CNS pathology, abnormal renal or hepatic function, history of malignancy other than ALL, uncontrolled infection, clinically relevant coronary artery disease or other relevant disease were also excluded.
The study was an open-label, dose-escalation study that followed a modified accelerated-titration design until DLT was observed or two patients exhibited Grade 2 non-haematological toxicity related to the study drug during Cycle 1 (12). The study then switched to a standard escalation method with 3–6 patients per cohort. In this study, DLT was defined as any Grade 3 or higher non-haematological toxicity (with the exception of asymptomatic abnormal laboratory abnormalities returning to initial levels within 7 days, fever/infection manageable with antibiotics and fatigue) or any treatment delay ≥7 days due to a drug-related AE.
AFM11 was given as a continuous infusion over 2 weeks, with a dose titration step from Week 1 to Week 2. This schedule followed the same approach as established for blinatumomab in ALL where it has been shown to reduce the risk of cytokine release syndrome and neurotoxicity (14, 15). The starting dose of AFM11 in Cohort 1 was 0.0007 µg/kg/week for Week 1 increasing to 0.002 µg/kg/week for Week 2. Dose escalation followed a modified Fibonacci regimen, with escalation of a half-log from the initial dose through Cohort 4 and reduced increments (100%, 67%, 50%, 30–35%) from Cohort 5.
In patients with a high tumour burden (e.g., more than 50% blasts, or more than 15,000 blasts/µL blood, or elevated lactate dehydrogenase more than twice the upper limit of normal), pre-treatment with 10 mg/m2 dexamethasone and 200 mg cyclophosphamide for up to 5 days was permitted. No immunosuppressive agents were allowed 4 weeks prior to and during AFM11 therapy; no other investigational agents were permitted during the study. Any medication necessary for the patient’s safety and well-being was given at the discretion of the investigator. Non-steroidal antiinflammatory drugs were permitted for a fever of ≥37.5°C, with the addition of dexamethasone for a fever of ≥38.5°C.
Safety was assessed by clinical review of all relevant parameters, including AEs, clinical laboratory evaluations (chemistry, haematology, coagulation analysis, urinalysis, and detection of tumour lysis syndrome), vital signs, physical examination (including ECOG performance status), cardiac monitoring, and neurological assessments. The number of patients with DLTs was summarised by severity, relatedness and preferred term/system organ class.
Efficacy was determined using bone marrow assessment performed at baseline and during evaluation visits during each cycle (Day 15–18). Minimal residual disease diagnostic tests were performed at baseline and at each evaluation visit by a central laboratory.
Blood samples were taken for PK analysis, biomarker and FACS analysis at regular intervals after the start of the infusion in both Weeks 1 and 2. Blood samples were analysed by flow cytometry for peripheral B-cell and T-cell depletion. Serum biomarker assessments were performed at a central laboratory. Immunogenicity was assessed by analysis of the presence of ADAs using an enzymelinked immunosorbent assay.
The safety population included all patients receiving at least one dose of study drug. The DLT analysis set included all patients who received ≥80% of the assigned dose and completed the DLT observation period or discontinued due to a DLT during the DLT observation period.