Effect of change in pI produced by chelator conjugation to mAb on tumor and organ uptake of In-111 labeled mAb

Objectives To investigate the effect of the amount of chelator conjugated to the antibody on the isoelectric point (pI), immunoreactivity, and biodistribution of the mAb.

Methods MORAb-009, a mAb directed against mesothelin, was conjugated with CHX-A”-DTPA (Macrocyclic, Inc), 2-(p-isothiocyanato-benzyl)-cyclohexyl-diethylenetriamine-pentaacetic acid, at a molar excess. The level of conjugation was determined by the Yttrium/Arsenazo III method for the CHX-A” concentration and the Bradford assay for the MORAb-009 concentration. The CHX-A”- MORAb-009 conjugates were subjected to isoelectric focusing to determine the pIs. The conjugate with 2.4 CHX-A” molecules/MORAb-009 was then labeled with In-111, and subjected to cell-binding assays for immunoreactivity and to a biodistribution study in groups of nude mice (n = 5-10 mice/group) with mesothelin-expressing A431/K5 tumors. The mice received i.v. In-111-CHX-A”- MORAb-009 (2 μCi /30 μg mAb) when the mean tumor size reached < ~130 mm3. The mice were euthanized at 1, 2, and 5 days post-injection for biodistribution.

Results The pI decreased in proportion to the level of CHX-A” conjugation; the pI was centered at ~8.1, ~7.9, ~7.2, and ~7.0 for MORAb-009 without CHX-A”, a conjugate with 2.4 CHX-A”/ MORAb-009, 3.5 CHX-A”/ MORAb-009, and 5.5 CHX-A”/ MORAb-009, respectively. The immunoreactivity (n=3) was 64 ± 10% for 2.4 CHX-A”/ MORAb-009. The conjugate with 2.4 CHX-A” showed a high tumor uptake value (27.6 ± 7.1% ID/g), and a low liver (8.4 ± 2.2% ID/g) and spleen uptake value (11.4 ± 4.8% ID/g) at 1 day.

Conclusions The conjugation with 2.4 CHX-A” molecules/mAb maintained the pI above 7.4, resulting in a net negative charge for the conjugate at the physiological pH of 7.4 and a good immunoreactivity. This conjugate showed good tumor targeting pharmacokinetics in nude mice with < ~130 mm3 tumors and warrants further studies for use in radioimmunotherapy