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

Visual Abstract

Non-Hodgkin lymphoma (NHL) will be diagnosed in approximately 80,000 patients and cause over 20,000 deaths in the United States in 2023 (1). Although chemotherapy is initially effective, many patients, even with low-grade lymphomas, relapse (2). This has driven development of therapeutic antibodies that target the CD20 protein expressed on the surface of mature B cells and most NHL cells, as most are of B-cell origin. Anti-CD20 immunotherapy has a highly favorable safety profile, significantly improves the outcomes of most patients, and, along with chemotherapy, is now part of the standard of care for many cases of NHL.
The chimeric mouse-human monoclonal antibody, rituximab, was the first Food and Drug Administration-approved anti-CD20 therapeutic, with others subsequently developed for improved biologic and pharmacologic properties, including fully human ofatumumab (3). Ofatumumab binds CD20 with high affinity, allowing targeting of cells with low CD20 expression, including those with resistance to rituximab (4). As a type I anti-CD20 antibody, ofatumumab is effectively internalized (5), which benefits imaging and therapy using residualizing radiometals, such as 89 Zr and 225 Ac.
Lymphoma is highly susceptible to ionizing radiation; however, external-beam irradiation is used sparingly in the disseminated setting. To overcome the limitations of external-beam radiotherapy, systemically administered b-particle-emitting radioimmunotherapies to anti-CD20 have been translated into 2 Food and Drug Administration-approved drugs: murine 131 I-tositumomab (Bexxar; GlaxoSmithKline) and 90 Y-ibrutumomab (Zevalin; Acrotech Biopharma LLC) (6). Although studies show long-term safety and effectiveness (7), Bexxar has been discontinued commercially in the United States, and Zevalin is used infrequently (8,9). 225 Ac has gained increased use as a therapeutic radionuclide (10), with its 10-d half-life matching well the pharmacokinetics of intact antibodies. In its decay pathway, 225 Ac yields 4 net a-particles with high linear energy transfer and short pathlengths, providing effective radiotoxicity to targeted tumor cells while relatively sparing nontargeted cells.
We previously demonstrated that [ 89 Zr]Zr-ofatumumab has excellent uptake into human lymphoma xenografts and enables in vivo localization using PET as well as, or better than, [ 89 Zr]Zrrituximab (11). Here, we prepare [ 225 Ac]Ac-ofatumumab and demonstrate potent cytotoxicity to CD20-expressing cells in vitro and in vivo. In therapeutic studies using an aggressive, disseminated murine model of human lymphoma, [ 225 Ac]Ac-ofatumumab shows excellent, often curative, efficacy.

Reagents and Cell Lines
Chemicals and reagents are listed in Supplemental Table 1 (supplemental materials are available at http://jnm.snmjournals.org). Water (MilliQ Integral 5 system; Millipore) was treated with a 50 g/L concentration of Chelex 100 (Bio-Rad Laboratories, Inc.). Raji and Raji-Luc cells were cultured in RPMI medium with 10% fetal bovine serum.
DOTA Conjugation, 225 Ac Chelation, Radiochemical Yield, Purity, and Mass Spectrometry DOTA was dissolved in H 2 O and conjugated to antibodies as previously described (11). For 225 Ac chelation, 1.85 MBq of 225 Ac in 0.2 M HCl was added to 2 M tetraethyl-ammonium-acetate to obtain pH 6.0. One hundred micrograms of DOTA antibody prepared at an 8:1 DOTAto-antibody molar ratio were added, and the reaction was brought to 150 mL with 20 mM sodium acetate, 150 mM NaCl (pH 7.0), and 15 mL of a 10 mg/mL solution of sodium ascorbate. After 4 h at 37 C, diethylenetriaminepentaacetic acid (pH 7.0) was added to 5 mM final concentration for 10 min followed by size-exclusion column purification into saline with sodium ascorbate added to 10 mg/mL. All quantifications were at secular equilibrium, using a Capintec CRC 55tW dose calibrator and a Beckman 8000 g-counter with a 250-to 480-keV energy window or by scanning of thin-layer chromatography strips. Fast protein liquid chromatography, thin-layer chromatography, and mass spectrometry were performed as previously described (12).

Immunoreactivity, in Vitro Stability, and Cell Killing
Immunoreactivity was assayed by Raji-cell binding as previously described (11), without or with cold ofatumumab blocking. To assay stability, [ 225 Ac]Ac-ofatumumab or 225 Ac was added to human serum, incubated at 37 C, and assayed by thin-layer chromatography as previously described (11).

Biodistribution in Tumor-Bearing Mice
The Washington University in St. Louis Institutional Animal Care and Use Committee approved the animal studies. Eight-to 10-wk-old R2G2 mice (no. 021; Envigo) were inoculated subcutaneously with 5 3 10 6 Raji-Luc cells. When palpable tumors were present, 8-11 mg (4.07 kBq) of [ 225 Ac]Ac-ofatumumab were administered intravenously and biodistribution analyzed 7 d later. The femur was measured after marrow extraction.
Therapy and Bioluminescent Imaging R2G2 mice were injected intravenously with 1 3 10 6 Raji-Luc cells. In a first study, 8 d later, the mice either were untreated or were treated with ofatumumab or 3.7 or 9.25 kBq/mouse of [ 225 Ac]Ac-IgG or [ 225 Ac]Ac-ofatumumab. In a second study, 12 or 16 d after cell injection, the mice either were untreated or were treated with 9.25 kBq of [ 225 Ac]Ac-ofatumumab. The administered antibody mass was adjusted to 20 mg/mouse of IgG or ofatumumab. Bioluminescent images were acquired and quantified as previously described (12,13) and normalized to the first imaging time point. The mice were humanely euthanized if they had hind-limb paralysis (HLP), more than a 20% weight loss, or morbidity or reached the scheduled study termination point.

Biodistribution and Dosimetry in Wild-Type Mice
Biodistribution and dosimetry studies were performed on 6-to 8wk-old female C57BL/6N mice (no. 556; Charles River) injected intravenously with 3 mg ($3.7 kBq) of [ 225 Ac]Ac-ofatumumab. At selected time points after injection of [ 225 Ac]Ac-ofatumumab, the mice were euthanized and organs g-counted at secular equilibrium to determine decay-corrected percentage injected activity per gram (%IA/g). Bone (tibia and fibula) was counted after marrow separation.
Integrated time-activity curves from the murine data and the mean absorbed dose (D) were calculated according to MIRD methodology (14,15) using the formula D 5 A 3 D 3 f, where A is the cumulated activity, D is the mean a-particle energy, and f is the absorbed fraction, with extrapolation to infinity, yielding a maximally conservative estimate. a particles were assumed to deposit all their energy locally (f 5 1). The trapezoidal rule was used to integrate the time-activity curve of the a-particles emitted from the decay of 225 Ac and its a-particle-emitting daughters ( 221 Fr, 217 As, 213 Bi, and 213 Po) using values from International Commission on Radiological Protection publication 107 (16), with all daughter decays assumed to occur in the same organ as the 225 Ac decay, yielding a D of 4.42 212 J/(BqÁs) for 225 Ac and its daughters. These mouse data were extrapolated to the adult female human model using the relative organ mass scaling method. Equilibrium in the decay chain and no translocation during the decay between succeeding disintegrations were assumed. Thus, the same estimated integrated time-activity curve obtained for 225 Ac was applied to its daughters. The absorbed dose of [ 225 Ac]Ac-ofatumumab was summed after applying weighting factors in the 2 possible pathways, 2% for 209 Ti and 98% for 213 Po. A relative biological effectiveness of 5 for a-particles was used in the calculation of sieverts.

Statistical Methods
GraphPad Prism software, version 8.4.3, was used for statistical analyses. A P value of less than 0.05 was considered statistically significant, with statistical tests noted in the text or figure legends. Data are shown as mean 6 SD.

Biodistribution with Subcutaneous Raji-Cell Tumors
We used immunodeficient R2G2 mice (B6;129-Rag2 tm1Fwa II2rg tm1Rsky /DwlHsd) that are proficient in double-strand DNAbreak repair. Prkdc scid mice that lack double-strand DNA repair because of the scid mutation are known to show artifactual radiosensitivity to DNA damage (18), such as that induced by a-particles. Compared with Prkdc scid mice, we expect that the nontargeted (nontumor) cells in R2G2 mice will better reflect the response of nontargeted (nontumor) cells in humans to a-particle transit, as both tumor and nontumor cells are proficient in doublestrand DNA-break repair.
[ 225 Ac]Ac-ofatumumab human radiation dosimetry estimates were then determined for a human female model ( Table 1). As free 213 Bi in the kidney was not evaluated, this absorbed dose may be somewhat underestimated (21). Extrapolated human radiation dose estimates reveal heart wall (1,919 mSv/MBq) as the organ with the highest predicted dose, followed by liver, spleen, and red marrow at 1,833, 1,803, and 1,620 mSv/MBq, respectively. Marrow is likely the dose-limiting organ. The calculated effective dose equivalent was 1,496 mSv/MBq.

Therapeutic Evaluation in a Disseminated Model on Day 8 After Cell Injection
The magnitude, duration, and tumor targeting of the radiopharmaceutical in the biodistribution studies, as well as the in vitro tumoricidal activity, motivated an in vivo lymphoma treatment study. Therapeutic efficacy was evaluated in R2G2 mice with intravenously injected Raji-Luc cells, which become widely disseminated (13,22,23). This model recapitulates many features of clinical NHL, as it invades multiple organs, including the hematopoietic compartment. HLP is a frequent cause for censoring.   First, the maximal tolerated dose of [ 225 Ac]Ac-ofatumumab in naïve R2G2 mice was identified. Eighty days after injection, 3.7, 11.1, 18.5, and 37 kBq/mouse yielded 5 of 5, 4 of 5, 3 of 5, and 0 of 5 survivors, respectively. Thus, for therapy we used single injections of the nonmyeloablative doses of 3.7 kBq/mouse (low dose) and 9.25 kBq/mouse (high dose).
In the first therapy study, 8 d after cell injection the mice were randomized to remain untreated or to be treated with native ofatumumab or low or high doses of [ 225 Ac]Ac-IgG or [ 225 Ac]Acofatumumab (n 5 10 mice per cohort). Survival, tumor burden, and weight were monitored for 200 d. In untreated mice, median survival was 21 d, with 9 of 10 mice succumbing before 29 d ( Fig. 2A) and all mice showing increasing cancer-cell burden until censoring for HLP (Figs. 2B and 3). The lone surviving mouse never displayed cancer cells, suggesting an unsuccessful injection or engraftment. Native ofatumumab did not extend survival compared with untreated mice, with 10 of 10 succumbing before 29 d ( Fig. 2A) and all showing increasing cancer-cell burden until censoring for HLP (Figs. 2B and 3).
Control radiolabeled nonspecific antibody, [ 225 Ac]Ac-IgG, at low and high doses yielded median survivals of 20 and 24 d, respectively ( Fig. 2A), which did not differ significantly from untreated or native-ofatumumab-treated mice (Mantel-Cox, P . 0.05). All mice in the low-dose [ 225 Ac]Ac-IgG cohort succumbed before 31 d. Nine were censored for HLP, and 1 perished (Fig. 3). In the high-dose cohort, 6 mice were censored for HLP and 3 for weight loss, with 1 surviving mouse that likely had an unsuccessful injection or engraftment (Figs. 2 and 3). All nonsurviving mice in both cohorts showed continuous cancer-cell growth until they were euthanized (Figs. 2B and 3). In contrast, low-or high-dose [ 225 Ac]Acofatumumab treatment increased median survival to 190 d and more than 200 d (median survival was nondeterminable as there were ,50% deaths), respectively ( Fig. 2A), superior to survival of untreated or [ 225 Ac]Ac-IgG-treated mice (Mantel-Cox, P , 0.05). In the low-dose cohort, 5 of 10 mice survived. One mouse was censored for HLP, 2 for weight loss, and 1 for development of a leg tumor; 1 perished (Fig. 3). In the high-dose cohort, 9 of 10 mice survived, with 1 succumbing under anesthesia with no prior morbidity, weight loss, or detectable cancer cells ( Figs. 2A and 3), indicating a death unrelated to disease or treatment. The median survival with high-dose [ 225 Ac]Ac-ofatumumab was longer than that with low-dose [ 225 Ac]Ac-ofatumumab (Mantel-Cox, P , 0.05), indicating a dose-response effect.
All mice treated with low-or high-dose [ 225 Ac]Ac-ofatumumab that survived had effective cancer-cell suppression with no detectable Raji-Luc cells at study termination (Figs. 2B and 3). The 5 nonsurviving mice in the low-dose cohort showed effective cancercell suppression for many days ($50 d for 4 mice and $140 d for 1 mouse), followed by resumption of Raji-Luc cell proliferation, as indicated by increased bioluminescence (Figs.  2B and 3).
Because of its 10-d half-life, we wanted to determine how rapidly [ 225 Ac]Ac-ofatumumab treatment kills cancer cells. Comparison of bioluminescence from study day 3 to day 20 (5 d before and 12 d after starting therapy) revealed a continued increase in cancer-cell numbers in all cohorts except if treated with [ 225 Ac]Ac-ofatumumab (Fig. 4A)  rapidly kills cancer cells in vivo, the log of the bioluminescence values from 10 to 20 d after cell injection (2-12 d after starting therapy) was plotted. Comparison of the slopes of these lines (Fig. 4B) confirmed no effect of native ofatumumab or [ 225 Ac]Ac-IgG on cancer-cell proliferation. In contrast, both low-and high-dose [ 225 Ac]Ac-ofatumumab significantly reduced these line slopes, indicating rapid killing of cancer cells after initiation of targeted therapy.

Evaluation of Systemic Toxicity
To investigate systemic toxicity of [ 225 Ac]Ac-ofatumumab, animal weights in the therapy study initiated 8 d after cell injection were determined (Supplemental Fig. 2). Not surprisingly, most nonsurviving mice in all cohorts showed clear weight loss before they died or were euthanized for cause, consistent with the increased cancer-cell burden in these mice.
Mice in the low-dose [ 225 Ac]Ac-ofatumumab-treated cohort that survived to study termination showed a continuous gradual weight gain (Supplemental Fig. 2B, left). This was consistently slightly lower than the weight gain of control R2G2 mice that were not injected with cells or treated with therapy (Supplemental Fig. 2B), with a significant difference present only after 172 d (Supplemental Fig. 2B, right). Surviving mice in the highdose [ 225 Ac]Ac-ofatumumab cohort showed greater systemic effects, with initial loss of weight having a nadir at 27 d, followed by recovery to initial weight at 52 d and thereafter (Supplemental Fig. 2B).  Fig. 4).
In contrast, a single treatment with [ 225 Ac]Ac-ofatumumab 12 d after cell injection increased median survival to 40 d ( Fig. 5A; Mantel-Cox, P , 0.0001), although no mice survived beyond 41 d. This treatment prevented a further increase or yielded a reduction in cancer-cell burden (Figs. 5B and 5C; Supplemental Fig. 4C). In this cohort, 5 mice were censored for HLP and 1 for weight loss; 3 perished (Supplemental Fig. 4C).

DISCUSSION
Our studies further validate the use of anti-CD20 antibodies for radioimmunotherapy of NHL. We produced [ 225 Ac]Ac-ofatumumab with high immunoreactivity, radiochemical yield, and purity and with stable chelation of 225 Ac in vitro and in vivo. [ 225 Ac]Acofatumumab specifically killed CD20-expressing cells in vitro and showed high tumor uptake in vivo. In therapeutic studies using a murine model of disseminated human lymphoma, a single [ 225 Ac]Acofatumumab treatment showed excellent efficacy, with curative ability except for very advanced disease.  We prepared [ 225 Ac]Ac-ofatumumab using a mild 1-step procedure in which 225 Ac is directly chelated to DOTA-conjugated antibody at 37 C (17). Other investigators also have used this approach (24)(25)(26). 225 Ac was stably chelated, and uptake of [ 225 Ac]Ac-ofatumumab uptake by CD20-expressing subcutaneous tumors (28 %IA/ g) was similar to the 33 %IA/g of [ 89 Zr]Zr-DFO-ofatumumab (11). The approximately 5 MBq/mg specific activity obtained for [ 225 A]Ac-ofatumumab was sufficient for preclinical use and should enable scaling up for clinical use.
The major finding of the current study was the high therapeutic potency of [ 225 Ac]Ac-ofatumumab in mice with an aggressive murine model of disseminated human lymphoma, similar to micrometastatic disease in humans. When administered 8 d after cell injection, control unlabeled ofatumumab and low (3.7 kBq/mouse) or high (9.25 kBq/mouse) doses of nontargeted [ 225 Ac]Ac-IgG did not improve median survival compared with untreated mice or inhibit cancer-cell growth. In contrast, both low and high doses of [ 225 Ac]Acofatumumab rapidly and significantly inhibited cancer-cell growth and increased median survivals to 190 and more than 200 d, respectively. Half the mice in the low-dose group survived, and none of the animals in the high-dose group had disease-or treatment-related mortality. All mice in these 2 cohorts that survived until study termination showed apparent complete elimination of cancer cells, as no cancer cells were detected at study completion on day 200. Also, we tested the potential for systemic anticancer effects in disease settings of further progression. Twelve days after cell injection, high-dose [ 225 Ac]Ac-ofatumumab killed cancer cells and significantly extended median survival to 40 d, showing therapeutic benefit even with a high pretreatment burden of cancer cells, although the therapy was not curative. Not surprisingly, there was a limit to therapeutic efficacy; therapy initiated 16 d after cell injection was unable to improve survival. This lack of benefit was likely because approximately 3 d before expected death was not enough time to allow for sufficient targeting and absorbance of the dose of intact radiolabeled antibody, and possibly also because of altered pharmacokinetics due to the high cancer-cell burden (27).
Dosimetry of organs from [ 225 Ac]Ac-ofatumumab-injected C57BL/ 6N mice extrapolated to an adult female human model suggests that marrow will be the dose-limiting organ, as is common with unsealed, intact antibody-based radiotherapeutics, including Bexxar and Zevalin (6). It is worth noting that uptake in nontumor target organs can often be significantly reduced by predosing with unlabeled antibody, which improves lesion-to-background-organ ratios, as used in the Zevalin and Bexxar therapeutic regimens and in other work (28). Fully human ofatumumab is unlikely to induce an immune response, increasing the potential for fractionated dosing of [ 225 Ac]Ac-ofatumumab to ameliorate myelotoxicity. In addition, autologous stem-cell transplantation after radioimmunotherapy, as used in some Zevalin and Bexxar protocols, may be useful. Development of a theranostic partner, such as [ 89 Zr]Zr-ofatumumab (11), for personalized image-based dosimetry is also of interest. Of course, murine models of toxicity and therapeutic benefit are useful-but imperfect-indicators for treatment of human patients, because of multiple differences including short circulation times in mice and dose-volume geometric considerations. Determination of toxicity-therapeutic trade-offs will require careful evaluation in human trials.
Notably, low-dose [ 225 Ac]Ac-ofatumumab treatment 8 d after cell injection was curative in half the mice, which displayed weight gains similar to those of control naïve mice without tumor cells or therapy. The 5 nonsurviving mice in this cohort showed effective repression of cancer-cell growth for approximately 50-140 d and gains of weight over this time of remission. Thus, fractionated cycles of this low-dose therapy may induce cancer-cell elimination, as well as curative efficacy in even more mice while maintaining reduced toxicity. Mice treated with high-dose [ 225 Ac]Ac-ofatumumab showed reduced weight gains compared with naïve mice, indicating some systemic toxicity. Some of this may result from kidney distribution of the 225 Ac daughter radionuclide, 213 Bi (29), although this possibility remains to be confirmed. However, if so, there are approaches that may ameliorate such toxicity (30,31).
We recently tested the therapeutic efficacy of b-particle-emitting [ 177 ]Lu-ofatumumab using this murine disseminated Raji-Luc lymphoma model (12). When therapy was started 4 d after cell injection, 8  with 213 Bi (half-life [t 1 =2], 45.6 min; 1 net a-particle), applying a similar disseminated Raji-Luc model using Prkdc scid mice (23). Cancer-cell killing was effective, and cures were common when the single-dose treatment was started 4 d after injection of 1 3 10 6 Raji-Luc cells. However, single-dose [ 213 Bi]Bi-rituximab treatment was much less effective when initiated 7 d after cell injection, being markedly less effective than single-dose treatment with [ 225 Ac]Ac-ofatumumab initiated 8 d after cell injection as found in the current study, although in a somewhat different animal system. These results may in part relate to the decay rates of 213 Bi versus 225 Ac and the localization times for intact antibodies to tumor. However, if targets are readily accessible, 213 Bi can deliver a high dose rate, but if there exist groups of cells, or even solid tumors, with reduced antibody accessibility, sufficient penetration or dose deposition may not occur before radioactive decay, rendering the therapy less effective and yielding superior results with the longerlived 225 Ac. In this view, for antibody-mediated radioimmunotherapy for larger tumors, 225 Ac may be a more appropriate choice for a-particle-mediated therapy. Further evaluation of the comparative therapeutic and off-target effects of 213 Bi-versus 225 Ac-labeling for radioimmunotherapy with intact antibodies is of interest.
Other investigators have studied aemitters for radioimmunotherapy of NHL using preclinical models. Park et al. (32) found therapeutic benefit using a pretargeted approach with a 213 Bi-labeled anti-CD20 1F5(ScFv) 4 SA molecule. Similarly, [ 211 At]At-1F5 ( 211 At t 1 =2, 7.2 h; 1 net aparticle) was 80% curative 6 d after intravenous cell injection with supporting stem cell transplantation but only poorly effective on subcutaneous tumors (33), perhaps limited by radioactive decay before tumor penetration. [ 212 Pb]Pb-rituximab ( 212 Pb t 1 =2, 10.6 h; 1 net a-particle) in a disseminated model improved survival times with therapy initiated at both low-and high-tumor burdens (34). [ 227 Th]Th-rituximab ( 227 Th t 1 =2, 18.7 d; 5 net a-particles) was often curative of small subcutaneous tumors (35) and was superior to [ 90 Y]Y-ibritumomabtiuxetan, although targeting of the 223 Ra daughter to bone (36) adds complexity. Similarly, treatment with [ 149 Tb]Tb-rituximab ( 149 Tb t 1 =2, 4.2 h, 1 a-particle) shortly after intravenous injection of Daudi cells significantly increased survival (37). Our excellent results with 225 Ac labeling likely reflect a very good match between antibodyspecific targeting times to tumor and the half-life of this a-particle emitter. In conclusion, a single dose of [ 225 Ac]Ac-anti-CD20 ofatumumab showed excellent therapeutic efficacy in a murine model of human disseminated lymphoma and was often curative. Fractionated dosing may improve efficacy. With increasing use of targeted radiotherapies, a renewed application of radioimmunotherapy targeting CD20 for NHL seems warranted, given the exceptional therapeutic efficacy of [ 225 Ac]Acanti-CD20 ofatumumab and remaining unmet clinical needs in this disease.

CONCLUSION
[ 225 Ac]Ac-ofatumumab shows good in vitro characteristics and effectively targets CD20-expressing tumor xenografts. [ 225 Ac]Acofatumumab showed curative efficacy in a murine model of disseminated human lymphoma. DISCLOSURE Richard Wahl is on the scientific advisory board of Clarity Pharmaceuticals, Voximetry, and Seno Medical; has stock in Clarity Pharmaceuticals and stock options in Voximetry; receives honoraria from Bristol Myers Squibb, Actinium Pharmaceuticals, Jubilant Draximage, Siemens, Abderra, and ITM; and receives research support from Actinium Pharmaceuticals, BMS, Bayer, Siemens, and White Rabbit AI. Diane Abou and Daniel Thorek have an advisory board role for, and own stock in, Diaprost AB and Pharma15. This study was supported in part by the Radiological Society of North America (RR1646 to Mark Hoegger), National Institutes of Health (T32EB021955 to Richard