Evaluation of Radiolabeled Girentuximab In Vitro and In Vivo

Abstract

Girentuximab (cG250) targets carbonic anhydrase IX (CAIX), a protein which is expressed on the surface of most renal cancer cells (RCCs). cG250 labeled with ¹⁷⁷Lu has been used in clinical trials for radioimmunotherapy (RIT) of RCCs. In this work, an extensive characterization of the immunoconjugates allowed optimization of the labeling conditions with ¹⁷⁷Lu while maintaining immunoreactivity of cG250, which was then investigated in in vitro and in vivo experiments. cG250 was conjugated with S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (DOTA(SCN)) by using incubation times between 30 and 90 min and characterized by mass spectrometry. Immunoconjugates with five to ten DOTA(SCN) molecules per cG250 molecule were obtained. Conjugates with ratios less than six DOTA(SCN)/cG250 had higher in vitro antigen affinity, both pre- and postlabeling with ¹⁷⁷Lu. Radiochemical stability increased, in the presence of sodium ascorbate, which prevents radiolysis. The immunoreactivity of the radiolabeled cG250 tested by specific binding to SK-RC-52 cells decreased when the DOTA content per conjugate increased. The in vivo tumor uptake was < 10% ID/g and independent of the total amount of protein in the range between 5 and 100 µg cG250 per animal. Low tumor uptake was found to be due to significant necrotic areas and heterogeneous CAIX expression. In addition, low vascularity indicated relatively poor accessibility of the CAIX target.

Keywords: 177Lu-radiopharmaceuticals; carbonic anhydrase IX; girentuximab; radioimmunotherapy; renal cell carcinomas.

Figure 1

Conjugation of cG250 with DOTA via benzylthiocyano group: (a) Size exclusion-high performance liquid chromatography (SE-HPLC) chromatograms of different selected DOTA(SCN)-cG250 conjugates compared to the native cG250; (b) size exclusion-high performance liquid chromatography/ultraviolet (SE-HPLC/UV) chromatograms of DOTA(SCN)-cG250 conjugates prepared from the same batch; and (c) SDS-PAGE chromatograms of cG250-conjugates in reduced conditions.

Figure 2

Characterization of the DOTA(SCN)-cG250 conjugates by mass spectrometry: (a) Lysine occupancy in the LC (top) and HC of DOTA(SCN)-cG250 post trypsin in-gel digestion of the proteins (n = 3) in conjugate C2 (90 min), C3, C4, C5 (60 min), and C7 (30 min) and (b) mass measurements by MALDI-TOF MS of native cG250 and conjugates C3 and C7.

Figure 3

Immunoreactivity of DOTA(SCN)-cG250 conjugates with SK-RC-52 cells in vitro and in tumor tissue. (a) Concentration-dependent binding of native cG250 to SK-RC-52 cells and SK-RC-18 (control), as assessed by flow cytometry. The red dashed line corresponds to the IC50. (b) Flow cytometric assessment of CAIX recognition on SK-RC-52 cells of immunoconjugates C2 (90 min), C3 (60 min) and C7 (30 min). (c) Flow cytometric assessment of CAIX recognition on SK-RC-52 cells of immunoconjugates C3, C4, and C5 (60 min). (d) CAIX immunostaining in frozen SK-RC-52 tumor samples using native cG250 mAb and immunoconjugates C2, C3, C4, C5, and C7. CAIX Abcam (ab15086) as a positive control and Dako (P0214) as a negative control. 100 μm scale bar and 10X objective.

Figure 4

In vitro stability of [¹⁷⁷Lu]DOTA(SCN)-cG250 in the presence of NaAsc by TLC: (a) C3 radioconstructs at different activity concentrations without and with NaAsc respectively and (b) C2, C3, and C7 radioconstructs (2 MBq/µg) in human serum and HSA 20% in the presence of NaAsc.

Figure 5

Radioimmunoactivity in vitro of [¹⁷⁷Lu]DOTA(SCN)-cG250 to SK-RC-52 cells (n = 3). (a) Concentration-dependent binding of [¹⁷⁷Lu]DOTA(SCN)-cG250 using the conjugate C3 (60 min). (b) Recognition of radioconstructs from conjugates C2 (90 min), C3 (60 min) and C7 (30 min) to SK-RC-52 cells by radioimmunoassay at 2 MBq/µg specific activity. (c) Radioimmunoactivity in HSA 20%, HS and PBS at 2 MBq/µg specific activity using the conjugate C3. (d) Radioimmunoactivity of [¹⁷⁷Lu]DOTA(SCN)-cG250 at different activity concentrations using the conjugate C3. Blocking studies were performed with 50 µg of native cG250.

Figure 6

Biodistribution of [¹⁷⁷Lu]DOTA(SCN)-cG250 from conjugate C7 at 48 h using total protein dose adjusted between 5 and 100 µg and 12 MBq per animal (n = 4). (a) Organs: tumor, blood, spleen and liver. (b) Activity balance.

Figure 7

Relationship between tumor uptake and tumor volume per animal of the biodistribution of [¹⁷⁷Lu]DOTA(SCN)-cG250 from conjugate C7 at 48 h using total protein dose adjusted between 5 and 100 µg and 12 MBq per animal (n = 4).

Figure 8

Biodistribution of [¹⁷⁷Lu]DOTA(SCN)-cG250 from conjugate C7 using a 0.5 µg (2 MBq) and a 5 µg (18 MBq) protein dose per animal (n = 4).

Figure 9

Evaluation of vascular density and hypoxia in FFPE from four different SK-RC-52 tumors: (a–d) (H&E) staining; (e–h) CD31 immunostaining; and (i–l) HIF1α staining. (a) 200 µm scale bar, 5x objective. (b–d) 1000 µm scale bar, 1.5x objective; (inset) 50 µm scale bar, 20x objective.