Increasing literature suggests that cell adhesion molecule α4β1 integrin plays a pivotal role in autoimmune diseases and cancer development. Noninvasive visualization of α4β1 integrin in vivo will facilitate the understanding of its involvement in disease progression and development of targeted therapies. Due to lack of high-affinity targeting ligands, in vivo imaging of α4β1 integrin is much less explored than that of integrins αvβ3 and αvβ5. One-bead one-compound (OBOC) library approach offers an important tool to identify novel ligands. Recently, we have developed topologically segregated bilayer bead encoding methods for OBOC libraries, thus the diversity of OBOC combinatorial libraries can now be substantially increased, and there are many more options for library design. These developments enable us to fully use the power of OBOC libraries for the identification of cancer-targeting agents having both peptidic and nonpeptidic features.

Here we described both the strategy of combining diverse 'initial' and highly 'focused' OBOC combinatorial libraries and the ways this strategy generated a high-affinity and highly specific peptidomimetic ligand to a4bl integrin. In conjunction with a high stringency screening method, we identified a high-affinity (IC₅₀=2 pM) peptidomimetic ligand (LLP2A) against α4β1 integrin using this strategy. LLP2A binds to the activated form of α4β1 integrin preferentially. We demonstrated that, when biotinylated and complexed with near infrared dye Alexa680-streptavidin conjugate to form a tetravalent molecule, LLP2A was able to target human lymphoma xenograft in nude mice with high sensitivity and specificity.

Although it is convenient to use streptavidin-fluorochrome as a contrast agent to evaluate in vivo targeting potential of LLP2A, this protein-based imaging probe loses the advantages of LLP2A being small molecule-based imaging probes, such as, fast kinetics and strong tumor penetration ability. We further demonstrated the use of small molecule imaging probe, LLP2A-Cy5.5 conjugate, as an in vivo near infrared optical imaging agent for lymphoma in a xenograft model. The subcutaneous Molt-4 tumor was clearly visualized from 0.5 h to 24 h after tail vein injection of the conjugate LLP2A-Cy5.5. Direct imaging and confocal microscopic examination of excised tumors and organs after probe injection confirmed the accumulation of LLP2A in tumors and revealed very little or no uptake in normal organs except for lymph nodes. Thus LLP2A has been shown great potential as an imaging probe to noninvasively monitor α4β1 expression and activity during tumor progression.