111In-LLP2A-DOTA Polyethylene Glycol-Targeting α4β1 Integrin: Comparative Pharmacokinetics for Imaging and Therapy of Lymphoid Malignancies

N-[[4-[[[(2-ethylphenyl)amino]carbonyl]amino]phenyl]acetyl]-N-epsilon-6-[(2E)-1-oxo-3-(3-pyridinyl-2-propenyl)]-L-lysyl-L-2-aminohexanedioyl-(1-amino-1-cyclohexane)carboxamide (LLP2A) is a high-affinity, high-specificity peptidomimetic ligand (inhibitory concentration of 50% 5 2 pM) that binds the activated alpha 4 beta 1 integrin found on a variety of malignant lymphoid cell lines. To better determine whether this ligand holds promise for imaging and therapy in lymphoid malignancies, 6 LLP2A derivatives, as LLP2A-1,4,7,10-tetraazacyclododecane-N,N',N '',N'''-tetraacetic acid (LLP2A-DOTA) and LLP2A-DOTA-polyethylene glycol (LLP2A-DOTA-PEG), were designed, synthesized, and radiolabeled with In-111. Comparative pharmacokinetic studies in mice with Raji B-cell lymphoma xenografts were then complemented by small-animal PET of the lead molecular LLP2A format using Cu-64-LLP2A-11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (Cu-64-LLP2A-CB-TE2A). Methods: LLP2A-DOTA and LLP2A-CB-TE2A were prepared using solid-phase synthesis; LLP2A-DOTA-PEG(2,000), LLP2A-DOTA-PEG(5,000), LLP2A-DOTA-PEG(10,000), (LLP2A-DOTA)(2)PEG(10,000), and (LLP2A-DOTA)(4)PEG(10,000) were prepared by PEGylation. In-111 radiolabeling of DOTA and Cu-64 radiolabeling of CB-TE2A conjugates yielded 370-1,850 and 3,700-7,400 kBq/mu g (10-50 and 100-200 mu Ci/mu g), respectively. The pharmacokinetics of the six In-111 radioconjugates were studied in vivo using biodistribution data (4 and 24 h) and whole-body autoradiography (24 h) in mice with Raji tumor xenografts. Cu-64-LLP2A-CB-TE2A was imaged (4 and 24 h) on a small-animal PET scanner in the same mouse model. Results: The highest tumor uptake in pharmacokinetic studies was obtained with LLP2A-DOTA and (LLP2A-DOTA)(4)-PEG(10,000). For In-111-LLP2A-DOTA (1 nM) at 4 and 24 h after injection, ratios of tumor to blood and tumor to nontumor (normal) organ (T/NT) were 8 to 35:1 for all organs or tissue except the spleen, marrow, and kidney, which were between 2:1 and 1:1. Tetravalent (LLP2A- DOTA)(4)PEG(10,000) (1.1 nM) had tumor uptake similar to the univalent LLP2A- DOTA but higher liver, marrow, and kidney uptake. The excellent T/NT of LLP2A was also demonstrated by small-animal PET with Cu-64-LLP2A-CB-TE2A at both 4 and 24 h after injection; obvious spleen targeting was apparent, but little kidney or liver activity was observed. Conclusion: Of the conjugates investigated, the univalent, non-PEGylated ligand In-111-LLP2A-DOTA exhibited the best T/NT ratios and showed the greatest potential for imaging of alpha 4 beta 1 in human lymphoma. Furthermore, this univalent non-PEGylated LLP2A format, as Cu-64-LLP2A-CB-TE2A, demonstrated excellent tumor targeting by small-animal PET and warrants further investigation as an agent for the study of alpha 4 beta 1 expression in human lymphoid malignancies.