This was a single-centre open-label, dose-escalation Phase I study (ClinicalTrials.gov identifier NCT01212887) managed and conducted in accordance with the principles of Good Clinical Practice and UK legislative requirements (Medicines and Healthcare Regulatory Agency).
Primary objectives were to evaluate the feasibility of MFEζ CAR T cell therapy in patients with CEACAM5+ tumours, to assess toxicity and to determine dose of MFEζ CAR T cells for optimal survival in the circulation. Secondary objectives were to assess functionality of MFEζ CAR T cells isolated from the circulation, to obtain preliminary evidence of radiological response and to evaluate safety.
The trial was based on a 3 + 3 design for cohorts 1 to 3 and 4 + 3 for cohorts 4 and 5 (Table 2). If a dose-limiting toxicity was experienced, the cohort was to be expanded to six patients. All patients received pre-conditioning chemotherapy, MFEζ T cells then intravenous IL2 therapy. Patients in cohorts 1–3 received fludarabine chemotherapy (25 mg/m2/day for 5 days) with inter-cohort escalation of MFEζ T cells. Patients in cohort 4 received maximum MFEζ T cell dose with cyclophosphamide (60 mg/kg/day for 2 days) prior to fludarabine (25 mg/m2/day for 5 days) chemotherapy. All patients received IV IL2 (600,000 IU/Kg 15-min infusion every 8 h maximum 12 doses). IL2 was commenced 90 min after MFEζ T cells. Criteria for IL2 dose delay, reduction or discontinuation defined within the protocol resulted in administration of a variable number of IL2 doses.
Inclusion criteria for this study included patients with advanced, histologically confirmed CEACAM5+ malignancy where standard curative or palliative measures were not applicable, ≥18 years old, life expectancy over 3 months, performance status of 0 or 1, adequate renal, cardiac, haematological and biochemical function. Exclusion criteria included anti-cancer systemic treatment or radiotherapy within four weeks, on-going significant toxicity from previous therapies, brain metastases, significant non-malignant disease (including autoimmune disease), prior BMT, previous extensive radiotherapy, current other malignancies and patients taking, or likely to require systemic steroids or other immunosuppressants.
Adverse event (AE) monitoring commenced from the point of written consent. AEs were reported as per Common Terminology Criteria for Adverse Events (CTCAE) Version 3.0. The following dose-limiting toxicities were defined when they were almost certainly or probably drug related; toxicity ≥grade 3 as a result of MFEζ T cells; toxicity caused by MFEζ T cells or chemotherapy preventing commencement of IL2 within 24 h; toxicity ≥3 during IL2 therapy that did not resolve to ≤grade 2 within 48 h of stopping IL2; toxicity ≥grade 3 as a result of chemotherapy despite optimal supportive medication excluding bone marrow suppression.
Patients were treated as inpatients and discharged home when clinically appropriate. They were followed up as outpatients and underwent computerized tomography (CT) scans at 6 weeks, 3, 6 and 12 months which were reported to RECIST version 1.0.
Production of MFEζ CAR T cells
MFEζ CAR T cells were produced in compliance with Good Manufacturing Practice as previously described .
Blood collection, processing and cell counts
Blood samples were collected at pre-treatment, day 0 pre-infusion, 2, 6 h, days 1, 2, 3, 4 and 5 post infusion and weeks 1, 2, 3, 4, 5, 6, 12 and then 12 weekly until off trial. Within 24 h of blood draw, plasma and PBMCs were isolated from an EDTA blood at each time point following standard procedures and stored at −80 °C and in liquid nitrogen. An additional CPT™ Vacutainer tube [Becton–Dickinson (BD), NJ, USA] was collected at each time point for mononuclear cells isolation and gDNA extraction using a Wizard® Genomic DNA Purification Kit (Promega, WI, USA) following the manufacture’s protocol. Blood counts were collected daily during hospitalization and at each visit using a certified clinical haematology service. All sample processing and subsequent assays were performed in compliance with good clinical laboratory practice guidelines and subjected to independent quality assurance control.
Real-time PCR quantification of transduced cells
A validated quantitative PCR assay (qPCR) was developed to quantify the level of MFEζ CAR T cells in patient samples. A CAR-specific qPCR amplicon (MFEζ F primer 5′-CTTATTACTGCCAGCAAAGGAGTAGTT, R primer 5′-CAAAGCTCGCTCCGTCTGTAG, probe FAM-5′-CCCACTCACGTTCGGTGCTGGC) and genomic standard qPCR amplicon (b2 M, F primer 5′-GGAATTGATTTGGGAGAGCATC, R primer 5′-CAGGTCCTGGCTCTACAATTTACTAA, probe FAM-5′-AGTGTGACTGGGCAGATCATCCACCTTC) were used to determine total genome copies (b2 M) and transduced genome copies (MFEζ) per sample.
The assay was validated using a standard curve generated from a single-cell-cloned Jurkat-MFEζ cell line (100%) diluted to 10, 1, and 0.1% with non-transduced Jurkat gDNA. Each assay included a positive control of known transduction level (4%) and a non-transduced (0%) negative control. The acceptance criteria for the qPCR assay were set as Standard Curve R
2 value ≥0.95, positive control = 4% (±2%) and lower limit of detection = 0.1% transduced cells.
IFNγ ELISA analysis
A 96-well ELISA plate was coated for 2 h at 37 °C or overnight at 4 °C with 1 µg/ml IFNγ capture antibody (MAB-285, R&D systems, MN, USA) and then washed with PBS + 0.05% Tween. IFNγ standards were then added (200–0.5 pg/ml) along with 10 and 100 µl patient plasma for each sample. Following incubation at 37 °C for 1 h, 100 µl of biotinylated IFNγ detection antibody (BAF-285, R&D systems, MN, USA) was added to each well for a further hour at 37 °C. After three washes, Streptavidin peroxidase (POD) conjugate (Roche, Basel, Switzerland) was then added and the plate incubated at 37 °C for 30 min and then POD blue substrate (Roche, Basel, Switzerland) added for 30 min. The reaction was stopped by the addition of H2SO4 and the plate read at 450 nm. The concentration of IFNγ was calculated using the standard curve.
Determination of cytokine concentrations in serum samples by Luminex bead array
Concentrations of plasma cytokines were measured using the Bio-Plex Pro™ Human Cytokine 17-plex Assay kit (Bio-Rad Laboratories Inc, California, USA). Reconstituted standards, cytokine-specific coupled beads, detection antibodies and 50 µl of thawed serum samples were combined according to manufacturer’s instructions and data acquired with the Bio-Plex™ 200 reader (Bio-Rad Laboratories Inc, California, USA). Data were analysed using Bio-Plex Manager™ software v6.0 (Bio-Rad Laboratories Inc, California, USA).
Anti-mouse scFv assay
96-well microtiter plates were coated with MFE antibody (1 µg/ml in carbonate-bicarbonate buffer, 100 µl/well) for one hour at room temperature, washed with PBS and blocked with 5% Marvel/PBS/Tween solution (150 µl per well). After washing, the wells with incubated with either positive, negative control, PBS or patient samples diluted in 1% Marvel/PBS/Tween solution (1/100 dilution, 100 µl/well) in four replicates for 1 h. The wells were washed and incubated with 100 µl per well of the appropriate secondary antibodies diluted in 1% Marvel/PBS/Tween solution. For the patient samples, rabbit anti-human IgG was added for 1 h. The wells were washed and incubated with anti-rabbit HRP antibody (100 µl/well for 1 h). After washing, 100 µl/well substrate (o-phenylenediamine in phosphate citrate buffer) was added and the reaction stopped after 5 min by adding 4 M hydrochloric acid (50 µl/well). The OD of the wells was obtained at 490 nm. The results were recorded as positive or negative according to previously set criteria for human anti-MFE antibody assay.
CEACAM5 expression in the lung
Following discontinuation of the clinical trial, we assessed whether the observed respiratory symptoms could have been attributable to CEACAM5 expression in the lungs of the patients. We accessed nine lung tissue samples from Manchester Cancer Research Centre Biobank. Eight were non-cancerous tissue from patients undergoing resection for lung cancer and the ninth was from a patient with metastatic colorectal cancer undergoing a lung resection. IHC using the Col-1 antibody  and qPCR were used to explore CEACAM5+ expression in these samples.