cGMP production of astatine-211-labeled anti-CD45 antibodies for use in allogeneic hematopoietic cell transplantation for treatment of advanced hematopoietic malignancies

Abstract

The objective of this study was to translate reaction conditions and quality control methods used for production of an astatine-211(211At)-labeled anti-CD45 monoclonal antibody (MAb) conjugate, 211At-BC8-B10, from the laboratory setting to cGMP production. Five separate materials were produced in the preparation of 211At-BC8-B10: (1) p-isothiocyanato-phenethyl-closo-decaborate(2-) (B10-NCS), (2) anti-CD45 MAb, BC8, (3) BC8-B10 MAb conjugate, (4) [211At]NaAt, and (5) 211At-BC8-B10. The 211At-labeling reagent, B10-NCS, was synthesized as previously reported. BC8 was produced, then conjugated with B10-NCS under cGMP conditions to form BC8-B10. [211At]NaAt was produced by α-irradiation of Bi targets, followed by isolation of the 211At using a "wet chemistry" method. The clinical product, 211At-BC8-B10, was prepared by reacting [211At]NaAt with BC8-B10 in NH4OAc buffer (pH 5.5) for 2 min at room temperature, followed by size-exclusion chromatography purification. Quality control tests conducted on the 211At-BC8-B10 included evaluations for purity and identity, as well as pyrogen and sterility tests. Stability of the 211At-BC8-B10 in 25 mg/mL sodium ascorbate solution was evaluated at 1, 2, 4, 6 and 21 h post isolation. For qualification, three consecutive 211At-BC8-B10 clinical preparations were successfully conducted in the cGMP suite, and an additional cGMP clinical preparation was carried out to validate each step required to deliver 211At-BC8-B10 to a patient. These cGMP preparations provided 0.80-1.28 Gbq (21.5-34.5 mCi) of 211At-BC8-B10 with radiochemical purity of >97%. The preparations were found to be sterile and have a pyrogen level <0.50 EU/mL. Cell binding was retained by the 211At-BC8-B10. 211At-BC8-B10 in ascorbic acid solution demonstrated a radiochemical stability of >95% for up to 21 h at room temperature. The experiments conducted have defined conditions for translation of 211At-BC8-B10 production from the laboratory to cGMP suite. This study has allowed the initiation of a phase I/II clinical trial using 211At-BC8-B10 (NCT03128034).

Fig 1. Scheme depicting the five production steps in the cGMP production of ²¹¹At-BC8-B10.

Each of the products were isolated, purified and characterized. The product from each step had to meet preset release criteria prior to being used in the next step.

Fig 2. Picture showing setup and transfer syringes used for production and purification of ²¹¹At-BC8-B10.

Reagent vials containing (1) ammonium acetate, (2) BC8-B10, (3) sodium thiosulfate, and (4) sodium ascorbate are not shown. Those reagents were added via syringe to the reaction vial containing ²¹¹At. Once the additions were made, the reaction solution was taken up into the large syringe and 1/3 volume was added to the top of each column. Following that the pre-rinsed columns were sequentially eluted with 4 mL of sterile PBS/sodium ascorbate solution. Fractions containing the ²¹¹At-BC8-B10 were collected in the product vessel and other elution volumes were directed to the waste vessel.

Fig 3. FACS histograms of ²¹¹At-BC8-B10, BC8-B10 and BHV1 (isotype matched control) binding to Ramos cells.

Duplicate samples of ²¹¹At-BC8-B10 were obtained from three production runs used to qualify the radiolabeling process. The ratio of mean fluorescence intensity (MFI) values for the ²¹¹At-BC8-B10 to BC8-B10 provides information on how the antibody binding has been affected by the radiolabeling process. A value of >50% MFI by flow cytometry for CD45 positive cell binding of the ²¹¹At-BC8-B10 relative to non-labeled BC8-B10 is used as the cutoff for release using this assay. This value has been used previously in clinical trials with ¹³¹I-labeled BC8.

Fig 4. FACS binding histograms for four different ²¹¹At-labeled BC8-B10 preparations (containing ascorbic acid) after incubating 4 h at room temperature.

Fig 5. Bar graph showing Ramos cell binding for ²¹¹At-BC8-B10 after being kept at room temperature for 4 h.

¹²⁵I-labeled BC8-B10 was used as a reference standard and ¹²⁵I-labeled BHV-1 was used as an isotype-matched non-binding control.