Cure of Disseminated Human Lymphoma with [225Ac]Ac-Ofatumumab in a Preclinical Model

Immunotherapies that target the CD20 protein expressed on most non-Hodgkin lymphoma cells have improved clinical outcomes, but relapse is common. We prepared 225Ac-labeled anti-CD20 ofatumu-mab and evaluated its in vitro characteristics and therapeutic efficacy in a murine model of disseminated human lymphoma. Methods: 225Ac was chelated by DOTA-ofatumumab, and radiochemical yield, purity, immunoreactivity, stability, and chelate number were deter-mined. In vitro cell killing of CD20-positive, human B-cell lymphoma Raji-Luc cells was assayed. Biodistribution was determined as percentage injected activity per gram (%IA/g) in mice with subcutane-ous Raji-cell tumors (n = 4). [225Ac]Ac-ofatumumab biodistribution in C57BL/6N mice was performed to estimate projected human dosime-try. Therapeutic efficacy was tested in mice with systemically dissemi-nated Raji-Luc cells, tracking survival, bioluminescence, and animal weight for a targeted 200 d, with single-dose therapy initiated 8, 12, or 16 d after cell injection, comparing no treatment, ofatumumab, and low (3.7 kBq/mouse) and high (9.25 kBq/mouse) doses of [225Ac]Ac-IgG and [225Ac]Ac-ofatumumab (n = 8-10/cohort). Results: Radio-chemical yield and purity were 32% & PLUSMN; 9% and more than 95%, respectively. Specific activity was more than 5 MBq/mg. Immunoreac-tivity was preserved, and more than 90% of the 225Ac remained che-lated after 10 d in serum. Raji-Luc cell killing in vitro was significant, specific, and dose-dependent. In tumor-bearing mice, [225Ac]Ac-ofa-tumumab displayed low liver (7 %IA/g) and high tumor (28 %IA/g) uptake. Dosimetry estimates indicated that bone marrow is likely the dose-limiting organ. When therapy was initiated 8 d after cell injection, untreated mice and mice treated with cold ofatumumab or low-or high-dose [225Ac]Ac-IgG showed indistinguishable median survivals of 20-24 d, with extensive cancer-cell burden before death. Low-and high-dose [225Ac]Ac-ofatumumab profoundly (P< 0.05) extended median survival to 190 d and more than 200 d (median not determin-able), with 5 and 9 of 10 mice, respectively, surviving at study termina-tion with no detectable cancer cells. Surviving mice treated with high-dose [225Ac]Ac-ofatumumab showed reduced weight gain ver-sus naive mice. When therapy was initiated 12 d, but not 16 d, after cell injection, high-dose [225Ac]Ac-ofatumumab significantly extended median survival to 40 d but was not curative. Conclusion: In an aggressive disseminated tumor model, [225Ac]Ac-ofatumumab was effective at cancer-cell killing and curative when administered 8 d after cell injection. [225Ac]Ac-ofatumumab has substantial potential for clini- cal translation as a next-generation therapeutic for treatment of patients with non-Hodgkin lymphoma.