Targeted alpha therapy using a novel CD70 targeted thorium-227 conjugate in in vitro and in vivo models of renal cell carcinoma

Abstract

The cell surface receptor CD70 has been previously reported as a promising target for B-cell lymphomas and several solid cancers including renal cell carcinoma. We describe herein the characterization and efficacy of a novel CD70 targeted thorium-227 conjugate (CD70-TTC) comprising the combination of the three components, a CD70 targeting antibody, a chelator moiety and the short-range, high-energy alpha-emitting radionuclide thorium-227 (²²⁷Th). In vitro analysis demonstrated that the CD70-TTC retained binding affinity to its target and displayed potent and specific cytotoxicity compared to an isotype control-TTC. A biodistribution study in subcutaneous tumor-bearing nude mice using the human renal cell carcinoma cell line 786-O demonstrated significant uptake and retention with 122 ± 42% of the injected dose of ²²⁷Th per gram (% ID/g) remaining in the tumor seven days post dose administration compared to only 3% ID/g for the isotype control-TTC. Tumor accumulation correlated with a dose dependent and statistically significant inhibition in tumor growth compared to vehicle and isotype control-TTC groups at radioactivity doses as low as 50 kBq/kg. The CD70-TTC was well tolerated as evidenced by only modest changes in hematology and normal gain in body weight of the mice. To our knowledge, this is the first report describing molecular targeting of CD70 expressing tumors using a targeted alpha-therapy (TAT).

Keywords: alpha particles; radioimmunotherapy; renal cell carcinoma; targeted alpha therapy (TAT); thorium-227.

Figure 1. Determination of chelator to antibody ratio (CAR) by UV-size-exclusion chromatography

UV-absorbance for the CD70 antibody-chelator conjugate at 280 nm (A) and the UV-absorbance of the chelator within the CD70 antibody-chelator conjugate at 335 nm (B) were monitored in parallel.

Figure 2. Comparison of binding potencies in ELISA, flow cytometry and in vitro cytotoxicity

(A) ELISA on immobilized recombinant human CD70, comparing CD70 antibody, CD70 antibody-chelator conjugate and isotype control antibody-chelator conjugate. EC₅₀ values are given in nM. (B) Flow cytometry analysis on human 786-O cells, comparing CD70 antibody with CD70 antibody-chelator conjugate and an isotype control antibody-chelator conjugate. EC₅₀ values are given in nM. (C) In vitro cytotoxicity performed using CellTiter-Glo® assay for CD70-TTC and isotype control-TTC, radiolabeled at a specific activity of 50 kBq/μg, on 786-O cells at radioactive concentrations of 2 and 20 kBq/ml. At study days 3, 5 and 7, cell viability was measured and normalized to cells incubated in medium only. A non-radiolabeled CD70 antibody-chelator conjugate was included for comparison.

Figure 3. 786-O tumor sections from animals in the biodistribution study

Frozen tumor sections were prepared seven days after dose administration and analyzed for microvessel density (A), CD70-expression (B) and autoradiography (C) and (D). Alpha-tracks in “star-like formations” were observed in tumor sections treated with CD70-TTC (C), but not in tumors treated with isotype control-TTC (D).

Figure 4. Biodistribution of CD70-TTC in the subcutaneous 786-O xenograft model

Biodistribution of CD70-TTC and an isotype control-TTC was analyzed 7 days after single i.v. administration of each compound at a dose of 500 kBq/kg (protein dose of 0.36 mg/kg). (A) Accumulation of ²²⁷Th expressed in % of injected dose per gram, decay corrected to the timepoint of injection in tumors and organs. (B–C) Accumulation of ²²⁷Th (Bq/g) and ²²³Ra (Bq/g), decay corrected to timepoint of euthanization in tumors and organs.

Figure 5. Individual tumor growth curves in the subcutaneous 786-O xenograft model

At an average tumor size of 150 mm³, animals received a single i.v. injection (study day 15; indicated with an arrow). (A) Vehicle. (B) CD70 antibody-chelator conjugate. (C) Isotype control-TTC (500 kBq/kg at 0.36 mg/kg). (D–G) CD70-TTC (doses of 50, 100, 300 and 500 kBq/kg at 0.36 mg/kg). (H) Comparison of average tumor volumes at study day 103. Statistical analysis was performed using one-way ANOVA (Dunnett's testing) in comparison to vehicle (n.s., not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

Figure 6. Average tumor growth inhibition, survival analysis and body weight changes

Start of treatment is indicated with an arrow in each plot (study day 15). (A) Average tumor growth inhibition until study day 103. Statistical analysis was performed using one-way ANOVA (Dunnett's testing) in comparison to vehicle (n.s., not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. (B) Survival plot analysis of animals sacrificed due to humane endpoint (tumor volume > 1500 mm³). Animals were either treated with vehicle, CD70 antibody-chelator conjugate, isotype control-TTC (500 kBq/kg; 0.36 mg/kg) or with CD70-TTC at the indicated doses (50, 100, 300 or 500 kBq/kg; 0.36 mg/kg). The median survival time (MST) for the vehicle control group was determined to be 86 days and the MST of the CD70 antibody-chelator conjugate was 91 days, whereas the MST for animals treated with isotype control-TTC was 113 days. The MST for groups receiving CD70-TTC could not be determined. Data were analyzed using Log-Rank (Mantel-Cox) analysis (n.s., not significant; *, p < 0.05, **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). (C) Changes in body weight, expressed in %.

Figure 7. Hematology during course of in vivo efficacy study

Blood chemistry analysis was performed at study day 44, 65 and 114. Statistical analysis was performed using one-way ANOVA (Dunnett's testing) using vehicle as baseline (n.s., not significant; *, p < 0.05, **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). At day 114, no animals treated with isotype control-TTC were alive and therefore blood analysis was not applicable (n.a.). (A) White blood cell counts. (B) Lymphocytes. (C) Neutrophils. (D) Red blood cell. (E) Platelets.