Generation and in vivo characterization of a chimeric αvβ5-targeting antibody 14C5 and its derivatives

Abstract

Background: Previous studies showed that radiolabeled murine monoclonal antibody (mAb) 14C5 and its Fab and F(ab')2 fragments, targeting αvβ5 integrin, have promising properties for diagnostic and therapeutic applications in cancer. To diminish the risk of generating a human anti-mouse antibody response in patients, chimeric variants were created. The purpose of this study was to recombinantly produce chimeric antibody (chAb) derivatives of the murine mAb 14C5 and to evaluate the in vitro and in vivo characteristics.

Methods: In vitro stability, specificity, and affinity of radioiodinated chAb and fragments (Iodo-Gen method) were examined on high-expressing αvβ5 A549 lung tumor cells. In vivo biodistribution and pharmacokinetic characteristics were studied in A549 lung tumor-bearing Swiss Nu/Nu mice.

Results: Saturation binding experiments revealed high in vitro affinity of radioiodinated chAb, F(ab')2, and Fab, with dissociation constants (KD) of 1.19 ± 0.19, 0.68 ± 0.10, and 2.11 ± 0.58 nM, respectively. ChAb 14C5 showed highest tumor uptake (approximately 10%ID/g) at 24 h post injection, corresponding with other high-affinity Abs. ChF(ab')2 and chFab fragments showed faster clearance from the blood compared to the intact Ab.

Conclusions: The chimerization of mAb 14C5 and its fragments has no or negligible effect on the properties of the antibody. In vitro and in vivo properties show that the chAb 14C5 is promising for radioimmunotherapy, due to its high maximum tumor uptake and its long retention in the tumor. The chF(ab')2 fragment shows a similar receptor affinity and a faster blood clearance, causing less non-specific retention than the chAb. Due to their fast blood clearance, the fragments show high potential for radioimmunodiagnosis.

Figure 1

Purification of chAb and chF(ab')₂ 14C5 produced by transfected HEKT293T cells. Protein A elution of chAb 14C5 (a), gel filtration elution of chAb 14C5 (arrow indicates the chAb 14C5) (b), cation-exchange chromatography elution of chF(ab')₂ (c) and Hi-Trap elution of chF(ab')₂ (d). OD, optical density.

Figure 2

SDS-PAGE analysis of the produced chimeric antibody and fragments 14C5. Visualized using Coomassie brilliant blue dye (a) and specific visualization by Western blotting detected by mouse anti-human kappa antibody (lanes 2, 4, 5, and 6), mouse anti-His antibody (lane 3), followed by anti-mouse IgG1 alkaline phosphatase (b): lane 1, protein marker; lane 2, ch antibody; lane 3, enzymatically produced chF(ab')₂; lane 4, enzymatically produced chFab; lane 5, recombinant chF(ab')₂; lane 6, recombinant chFab (a, b). Phosphorescence image after SDS-PAGE: lane 1, estimated protein ladder based on non-radioactive protein marker; lane 2, ¹²⁵I-ch antibody; lane 3, ¹²⁵I-chFab; lane 4, ¹²⁵I-chF(ab')₂ 14C5 (c).

Figure 3

Saturation binding experiment of the chF(ab')₂ (triangle), ch antibody (square), and chFab (circle) 14C5. By cellular ELISA on A549 cells with mouse anti-human kappa antibody (IgG1) and anti-mouse IgG1-alkaline phosphatase. Data are expressed as mean ± SD (n = 3).

Figure 4

Saturation binding assay of iodinated derivatives of the mAb 14C5 to A549 tumor cells. Specific (circle) and non-specific binding (triangle) (binding to negative control Colo16 cell line) of ¹²⁵I-chAb 14C5 (a), of ¹²⁵I murine Ab 14C5 (b), of ¹²⁵I-chF(ab')₂ 14C5 (c), and of ¹²⁵I-chFab 14C5 (d). Data are expressed as mean ± SD (n = 3).

Figure 5

Blood clearance in NMRI mice and tumor uptake and tumor-to-blood ratio in A549 lung tumor-bearing mice. Blood clearance of ¹³¹I-labeled chFab, chF(ab')₂, and chAb 14C5 in NMRI mice (a). Tumor uptake (%ID/g) (b) and tumor-to-blood ratio (c) of ¹³¹I-labeled chAb, chF(ab')₂, and chFab 14C5 in A549 lung tumor-bearing mice. Values are corrected for decay of the radionuclide. Data are expressed as mean ± SD (n = 3 to 5).

Figure 6

Organ uptake of ¹³¹I-chFab, ¹³¹I-chF(ab')₂, and ¹³¹I-chAb 14C5. Organ uptake (%ID/g tissue) of ¹³¹I-chFab (a), ¹³¹I-chF(ab')₂ (b), and ¹³¹I-chAb 14C5 (c) in A549 lung tumor-bearing mice. Values are corrected for decay of the radionuclide. Data are expressed as mean ± SD (n = 3 to 5).

Figure 7

Tumor uptake and tumor-to-blood ratio of ¹³¹I-labeled chAb 14C5 and ¹³¹I-labeled ch MabThera. Tumor uptake (%ID/g) (a) and tumor-to-blood ratio (b) of ¹³¹I-labeled chAb 14C5 in A549 lung tumor-bearing mice and in Colo16-bearing mice and ¹³¹I-labeled ch MabThera in A549 lung tumor-bearing mice. Values are corrected for decay of the radionuclide. Data are expressed as mean ± SD (n = 3 to 5). The asterisk indicates p < 0.05, non-parametric Mann–Whitney U test.