A preclinical model of CD38-pretargeted radioimmunotherapy for plasma cell malignancies

Abstract

The vast majority of patients with plasma cell neoplasms die of progressive disease despite high response rates to novel agents. Malignant plasma cells are very radiosensitive, but the potential role of radioimmunotherapy (RIT) in the management of plasmacytomas and multiple myeloma has undergone only limited evaluation. Furthermore, CD38 has not been explored as a RIT target despite its uniform high expression on malignant plasma cells. In this report, both conventional RIT (directly radiolabeled antibody) and streptavidin-biotin pretargeted RIT (PRIT) directed against the CD38 antigen were assessed as approaches to deliver radiation doses sufficient for multiple myeloma cell eradication. PRIT demonstrated biodistributions that were markedly superior to conventional RIT. Tumor-to-blood ratios as high as 638:1 were seen 24 hours after PRIT, whereas ratios never exceeded 1:1 with conventional RIT. (90)Yttrium absorbed dose estimates demonstrated excellent target-to-normal organ ratios (6:1 for the kidney, lung, liver; 10:1 for the whole body). Objective remissions were observed within 7 days in 100% of the mice treated with doses ranging from 800 to 1,200 μCi of anti-CD38 pretargeted (90)Y-DOTA-biotin, including 100% complete remissions (no detectable tumor in treated mice compared with tumors that were 2,982% ± 2,834% of initial tumor volume in control animals) by day 23. Furthermore, 100% of animals bearing NCI-H929 multiple myeloma tumor xenografts treated with 800 μCi of anti-CD38 pretargeted (90)Y-DOTA-biotin achieved long-term myeloma-free survival (>70 days) compared with none (0%) of the control animals.

Figure 1

Components for pretargeted radioimmunotherapy (PRIT). Schema depicting (a) one-step conventional radioimmunotherapy and (b) multistep PRIT. PRIT involves infusion of the antibody-SA construct [Step 1], followed by injection of a synthetic N-acetylgalactosamine-containing clearing agent [Step 2] designed to facilitate hepatic clearance of excess antibody-SA from the bloodstream, and then infusion of the radiolabeled small molecule DOTA-biotin [Step 3]. (c) Schematic diagram of OKT10 scFv-SA fusion gene. (d) Size exclusion HPLC performed on OKT10-FP and OKT10-CC anti-CD38 streptavidin constructs demonstrate a retention time of 9.965 and 9.963 minutes respectively. A small amount of aggregate is present.

Figure 2

Binding of Anti-CD38 antibody constructs to CD38 on malignant plasma cells. Competitive binding assays demonstrate blocking of Alexa-647 conjugated OKT10 mAb binding to CD38-expressing L363 MM cells with escalating concentrations of unlabeled (a) OKT10-FP or (b) OKT10-CC. No blocking is observed with escalating concentrations of nonbinding control Ab constructs.

Figure 3

Biodistributions of radioactivity in athymic nude- Foxnl^nu mice (n=5/group) bearing CD38+ MM xenograft tumors (1×10⁷ cells injected into left flank). Blood, tumor and normal organ specimens were obtained 24hr after (a) ¹¹¹In-OKT10-DOTA or ¹¹¹In-BHV1-DOTA (control) (b) ¹¹¹In-DOTA-biotin pretargeted by OKT10-CC or BHV1-CC (control CC) (c) ¹¹¹In-DOTA-biotin pretargeted by OKT10-FP or CC49-FP (control FP). In PRIT animal studies (b,c) NAGB-clearing agent (5.8 nmol) was administered 2hr before the ¹¹¹In-DOTA-biotin (1.2 nmol). Comprehensive tissue biodistributions of radioactivity obtained at sequential timepoints 2hr, 24hr, 48hr and 96hr following ¹¹¹In-DOTA-biotin are shown for the mice from the OKT10-CC (d) and BHV1-CC (e) PRIT groups.

Figure 4

Effects of biotinylated N-acetyl galactosamine clearing agent (CA) on circulating OKT10-CC. CA [5.8 nmol] was injected 24hr after ¹³¹I-OKT10-CC (1.4 nmol) into 5 athymic nude mice (i.v.). Five control mice received ¹³¹I-OKT10-CC (1.4 nmol) and no CA. Retro-orbital venous sampling was conducted at serial time points up to 24 hr.

Figure 5

⁹⁰Y absorbed dose estimates (Gy/mCi) from an ¹¹¹In-DOTA-biotin tracer. Radiation absorbed doses to tumor, whole body and normal tissues are estimated by integrating the areas under time-activity curves constructed by plotting the concentration of ¹¹¹In-DOTA-biotin (after OKT10-CC pretargeting) measured in tissues by γ-counting at 2, 24, 48 and 96hr.(33)

Figure 6

Tumor responses in athymic nude-mice (n=8–10/group) bearing L363 (a, b) or NCI-H929 (c, d) right flank MM xenografts. Mice received 1.4 nmol (300 µg) of OKT10-CC or BHV1-CC (control) and CA [5.8 nmol (50 µg)]. Treatment groups received ⁹⁰Y-DOTA-biotin 24hr after the OKT10-CC at doses of 400 µCi (14.9 MBq), 800 µCi (29.6 MBq) & 1200 µCi (44.4MBq); and at doses of 800 µCi and 1200 µCi for the BHV1-CC control groups. (a) Mice were monitored thrice weekly for tumor volume measurements and were euthanized when tumors were ≥10% of body weight or when ulceration occurred as mandated by the institutional animal care committee. (Curves are truncated at the time the first animal was euthanized in each group). A dose dependent regression of MM tumor xenografts is demonstrated. (b) Kaplan-Meier analysis of cumulative survival of mice bearing L363 MM xenografts. (c) NCI-H929 treatment groups received 800 µCi (29.6 MBq) of ⁹⁰Y-DOTA-biotin 24hr after the OKT10-CC or BHV1-CC (control group). Complete regression of MM tumor xenografts in the OKT10-CC group is demonstrated. (d) Kaplan-Meier survival analysis of mice bearing NCI-H929 MM xenografts.

Figure 7

Tumor responses in athymic nude-mice (n=10/group) bearing L363 right flank MM xenografts. All mice received 1.4 nmol (300 µg) of OKT10-CC as a single agent (no radioactivity) or followed by CA [5.8 nmol (50 µg)] and ⁹⁰Y-DOTA-biotin (800 µCi [29.6 MBq]) 24hr after the OKT10-CC. (a) Mice were monitored thrice weekly for tumor volume measurements and were euthanized when tumors were ≥10% of body weight or when ulceration occurred as mandated by the institutional animal care committee. (Curves are truncated at the time the first animal was euthanized in each group). Complete regression of MM tumor xenografts in the group receiving OKT10-CC followed by 800 µCi (29.6 MBq) is demonstrated. Animals receiving OKT10-CC as a single agent (no radioactivity) demonstrated rapid tumor progression. (b) Kaplan-Meier analysis of cumulative survival of mice bearing L363 MM xenografts.