Preclinical Development of an Actinium-225-Labeled Antibody Radio-Conjugate Directed Against CD45 for Targeted Conditioning and Radioimmunotherapy

Introduction: Radiolabeled CD45 antibodies have demonstrated clinical promise as targeted conditioning treatments prior to bone marrow transplant (BMT) as a better tolerated and potentially more effective alternative to harsh chemotherapy and/or total body irradiation regimens for myeloablative conditioning. CD45 is expressed on all immune cells, including hematopoietic stem cells and both precursor and mature lymphoid and myeloid cells, with an average copy number up to 200,000 molecules per cell, making it an ideal antigen for directing targeted radiation for conditioning. It is also highly expressed on most lymphomas and leukemias, and approximately 50% of multiple myeloma, further offering the potential for mediating a substantial anti-tumor effect in treated patients. We describe the preclinical development of a novel anti-CD45 antibody radio-conjugate (ARC) that harnesses the power of alpha radiation by arming the pan-CD45 antibody BC8 with Actinium-225 (²²⁵Ac). ²²⁵Ac is a potent radionuclide with high linear energy transfer (80-100 keV/μm) yet short path length (50-80 μm) to deliver significantly more lethal double strand DNA breaks per alpha track than beta particles. In addition, the use of ²²⁵Ac for targeted conditioning has advantages of safe handling and ease of use and a long 10 day half-life that enables flexibility in preparation and use.

Methods and Results: To generate ²²⁵Ac-BC8, the monoclonal antibody was first conjugated with the DOTA chelator, then labeled with ²²⁵Ac, achieving a labeling efficiency typically greater than 80%. ²²⁵Ac-BC8 was subsequently purified to high radiochemical purity by column chromatography. Using human CD45⁺ Ramos lymphoma cell line and EL4 as negative control, ²²⁵Ac-BC8 was shown to be stable and retain selective immunoreactivity following conjugation and labeling. To evaluate the anti-tumor potency of ²²⁵Ac-BC8, two CD45⁺ human multiple myeloma (MM) cell lines with high or low relative CD45 antigen density, U266 and H929, respectively, (Collette, et al. 2007) were tested by cell cytotoxicity assay. Following transient exposure (4 or 12 hours) to ²²⁵Ac-BC8, the ARC demonstrated potent dose-dependent MM cell killing 72 hours post-treatment, achieving greater than 90% cytotoxicity of both cell lines. To further assess the therapeutic potential of ²²⁵Ac-BC8, its anti-tumor activity was tested in subcutaneous (s.c.) xenograft models of H929 and U266. NOD-SCID mice bearing established s.c. tumors were randomized to receive a single dose of 300 nCi of ²²⁵Ac-BC8, 300 nCi of an irrelevant radio-conjugate ²²⁵Ac-18B7, naked BC8 antibody, or no treatment control. In both models, ²²⁵Ac-BC8 exerted complete and durable tumor control for the duration of study. None of the control groups exhibited any anti-tumor effect. Studies to evaluate ²²⁵Ac-BC8 in models of CD45⁺ lymphoma are ongoing. Since BC8 does not cross-react with mouse CD45 and only weakly with macaque CD45, biodistribution, dosimetry and imaging analysis was performed in mice using radio-labeled anti-mouse pan-CD45 surrogate antibody 30F11. SPECT/CT imaging and biodistribution was performed with ¹¹¹In-labeled 30F11. Following IV administration, ¹¹¹In-CD45 (30F11) rapidly cleared from the blood and accumulated in immune system organs including spleen, bone marrow, and lymph node. Dosimetry calculations for ²²⁵Ac-CD45 (30F11) were determined for absorbed dose to target organs. Only modest uptake was noted in the kidney, but significant uptake occurred in liver, the major site for catabolism of full-length antibodies. ²²⁵Ac-CD45 (30F11) is a useful molecule to study the tolerability and myeloablative properties of ²²⁵Ac-CD45 antibody for targeted conditioning prior to donor BMT, using B6-Ly5^b donor cells in B6-Ly5^a mice. Results of ongoing studies will also be presented.

Conclusions: The pan-CD45 antibody BC8 was shown to be efficiently conjugated and labeled with the potent alpha emitter ²²⁵Ac to mediate robust and selective killing of CD45⁺ MM cells and xenografts. Biodistribution analysis confirmed selective uptake into organs of the immune system including homing to BM. Supported by ongoing myeloablative/BM reconstitution studies, these preclinical data support the development of ²²⁵Ac-BC8 as a novel potent and safe targeted conditioning approach for BMT.

Reference: Collette, et al., (2007). Eur. Cytokine Netw. 18:120-126.