In Vitro and In Vivo Comparison of Random versus Site-Specific Conjugation of Bifunctional Chelating Agents to the CD33-Binding Antibody for Use in Alpha- and Beta-Radioimmunotherapy

Abstract

Radiometal chelator conjugation is a cornerstone of radioimmunotherapy (RIT). Continued interest in selective placement of chelators remains an active topic of discussion in the field. With several simple site-specific methods being recently reported, it was of interest to investigate the benefits and potential drawbacks of the site-specific method with a full comparison to a more typical random conjugation method that is currently utilized in clinical applications. In this study, the conjugation methods were evaluated side by side to determine the utility of both methods using commercially available random and site-specific conjugation reagents by performing antigen binding; radiolabeling with ⁶⁴Cu, ¹⁷⁷Lu, and ²²⁵Ac radioisotopes; antibody-conjugate stability, cytotoxicity, in vivo distribution, pharmacokinetics analyses, and dosimetry to gather a whole data set for preclinical investigation. Evaluation revealed that both methods performed similarly during most experiments with the site-specific method, resulting in higher binding capacity of the antibody conjugate via flow cytometry. Radiolabeling was not significantly different between two methods, while stability showed that the site-specifically conjugated antibody was somewhat more stable at 37 °C in human serum over 1 week. In vitro experiments demonstrated less cell killing with the random conjugation method, while in vivo experiments showed no statistical differences in tumor uptake between conjugation methods. Dosimetry calculations were performed using the acquired PET/CT data and showed that apart from the liver, there was no significant difference in radiation doses delivered by either antibody conjugate. These results demonstrate that both methods are viable for future work, while the site-specific method offers several potential advantages and, in some cases, improved efficacy.

Figure 1

Conjugation of (A) nonspecific, p-SCN-Bn, and (B) site-specific, PFP, functionalized DOTA to an antibody molecule.

Figure 2

Immunoreactivity and binding data of modified HuM195 immunoconjugates. (A) Indirect enzyme linked immunosorbent assay (ELISA) of HuM195 immunoconjugates against recombinant human Siglec-3 (CD33). (B) Titration flow cytometry binding of HuM195 immunoconjugates to OCI-AML3 cells. (C) Comparison of the total binding capacity (Bmax) of immunoconjugates to that of naïve HuM195. (D) Binding curve of HuM195 immunoconjugates to Daudi cells as a negative control. Flow cytometry controls shown in Supporting Information Figure S1.

Figure 3

RadioHPLC and stability evaluation of radiolabeled PFP-HuM195 and SCN-HuM195 immunoconjugates. HPLC chromatograms run at a wavelength of 280 nm (blue) and radioactive trace (red). (A,B) ⁶⁴Cu-PFP-HuM195 and ⁶⁴Cu–SCN-HuM195; (C,D) ¹⁷⁷Lu-PFP-HuM195 and ¹⁷⁷Lu-SCN-HuM195; (E,F) ²²⁵Ac-PFP-HuM195 and ²²⁵Ac-SCN-HuM195; and (G) stability over 7 days for ²²⁵Ac-PFP-HuM195 and ²²⁵Ac-SCN-HuM195 in human serum and PBS.

Figure 4

Internalization into OCI-AML3 and in vitro cytotoxicity data of ²²⁵Ac-labeled HuM195 immunoconjugates and free ²²⁵Ac-DTPA control against OCI-AML3 cells. (A–C) Cytotoxicity curves for high (18.5 kBq), medium (4.63 kBq), and low dose (0.29 kBq) of ²²⁵Ac-labeled HuM195 immunoconjugates against CD33⁺ cells over 24 h. (D) 24 h dose–response curve of ²²⁵Ac-labeled HuM195 immunoconjugates against OCI-AML3 cells with calculated inhibitory constant (K_i) values. (E) Comparison of ²²⁵Ac-labeled HuM195 immunoconjugates effect on cell confluency compared to untreated control; and (F) internalization of HuM195 and of immunoconjugates into OCI-AML3 cells.

Figure 5

Representative micro-PET/CT images of OCI-AML3 tumor-bearing SCID female mice injected IV with (A) ⁶⁴Cu-PFP-HuM195 (unblocked), (B) ⁶⁴Cu-SCN-HuM195 (unblocked), (C) ⁶⁴Cu-PFP-HuM195 (blocked ), and (D) ⁶⁴Cu–SCN-HuM195 (blocked). Red arrows indicate tumor location. Green arrows indicate radiotracer joint uptake. All images displayed a maximum intensity projection (MIP). Unblocked mice were given only radiolabeled conjugates; blocked mice were given 0.5 mg HuM195 24 h prior to the radioactive dose.

Figure 6

Time–activity curves of the SOs in OCI-AML3 tumor-bearing SCID female mice injected IV with ⁶⁴Cu–SCN-HuM195 or ⁶⁴Cu-PFP-HuM195. The curves were obtained from standardized uptake values (SUVs) derived from micro-PET/CT images at different time points. The curves are presented in the following order: (A) blood clearance, (B) liver and intestine activity curves, (C) kidney and spleen activity curves, and (D) tumor kinetic curves.

Figure 7

Standardized uptake values normalized by body weight (SUVbw) in OCI-AML3 tumor-bearing SCID female mice injected IV with ⁶⁴Cu–SCN-HuM195 or ⁶⁴Cu-PFP-HuM195. Analysis of tumors was based on micro-PET/CT images taken at 1, 5, 20, 30, and 44 h post radioconjugate injection. Tumor region of interest (ROI) was drawn manually for SUV calculations. Significantly more uptake was seen in the unblocked mice starting at 20 h post injection relative to the blocked mice. *** and **** indicate p < 0.001 and p < 0.0001, respectively. Unblocked mice were given only radiolabeled conjugates; blocked mice were given 0.5 mg HuM195 24 h prior to the radioactive dose.