Design and validation of fibroblast activation protein alpha targeted imaging and therapeutic agents.

Background: Cancer-associated fibroblasts (CAFs) comprise a major cell type in the tumor microenvironment where they support tumor growth and survival by producing extracellular matrix, secreting immunosuppressive cytokines, releasing growth factors, and facilitating metastases. Because tumors with elevated CAFs are characterized by poorer prognosis, considerable effort is focused on developing methods to quantitate, suppress and/or eliminate CAFs. We exploit the elevated expression of fibroblast activation protein (FAP) on CAFs to target imaging and therapeutic agents selectively to these fibroblasts in solid tumors. Methods: FAP-targeted optical imaging, radioimaging, and chemotherapeutic agents were synthesized by conjugating FAP ligand (FL) to either a fluorescent dye, technetium-99m, or tubulysin B hydrazide. In vitro and in vivo studies were performed to determine the specificity and selectivity of each conjugate for FAP in vitro and in vivo. Results: FAP-targeted imaging and therapeutic conjugates showed high binding specificity and affinity in the low nanomolar range. Injection of FAP-targeted 99mTc into tumor-bearing mice enabled facile detection of tumor xenografts with little off-target uptake. Optical imaging of malignant lesions was also readily achieved following intravenous injection of FAP-targeted near-infrared fluorescent dye. Finally, systemic administration of a tubulysin B conjugate of FL promoted complete eradication of solid tumors with no evidence of gross toxicity to the animals. Conclusion: In view of the near absence of FAP on healthy cells, we conclude that targeting of FAP on cancer-associated fibroblasts can enable highly specific imaging and therapy of solid tumors.

Small molecule targeted NIR dye conjugate for imaging LHRH receptor positive cancers.

Overexpression of Luteinizing Hormone Releasing Hormone Receptor (LHRH-R) in various cancers and restricted expression of the receptor in healthy cells qualifies it as a valuable cancer biomarker. Previously, LHRH-R targeted peptides have been utilized to deliver attached payloads to LHRH-R expressing cancers. We report here for the first time the utilization of a small molecule non-peptidic ligand (BOEPL) of LHRH-R to deliver attached payloads to LHRH-R positive tumors. For this purpose, we linked the BOEPL ligand to a near infrared dye via various linkers. In vitro, these conjugates demonstrated low nanomolar binding affinity and in vivo they exhibited receptor-mediated uptake specifically in tumor tissue. Moreover, tumor uptake could be blocked by administration of excess unlabeled conjugate, and time course experiments showed retention of the dye conjugate in the tumor up to 12 h post injection. Because uptake of BOEPL-targeted NIR dye conjugates by nonmalignant organs/tissues was negligible and since the transient presence of targeted NIR dye in the kidneys was a result of clearance mechanism, we suggest that a BOEPL-targeted NIR dye might constitute a useful agent for fluorescence-guided surgery of LHRH-R positive cancers. Moreover, our results also provide proof of concept that BOEPL can be successfully used to deliver attached payloads to LHRH-R positive tumors in vivo.

Targeted Tubulysin B Hydrazide Conjugate for the Treatment of Luteinizing Hormone-Releasing Hormone Receptor-Positive Cancers.

The targeted delivery of chemotherapeutic agents to receptors that are overexpressed on cancer cells has become an attractive strategy to concentrate drugs in cancer cells while avoiding uptake by healthy cells. Luteinizing hormone-releasing hormone receptor (LHRH-R) has attracted considerable interest for this application, since LHRH-R is upregulated in ∼86% of prostate cancers, ∼80% of endometrial cancers, ∼80% of ovarian cancers, and ∼50% of breast cancers, but virtually absent from normal tissues. Although LHRH and related peptides have been used to deliver cytotoxic drugs to LHRH-R overexpressing cancer cells, they have suffered from off-target delivery of the therapeutic agents to the liver and kidneys. To reduce such unwanted uptake by peptide scavenger receptors in the liver and kidneys, we have explored the use of a nonpeptidic LHRH-R antagonist (NBI42902) to construct an LHRH-R targeted tubulysin conjugate (BOEPL-L3-TubBH). In vitro studies with BOEPL-L3-TubBH demonstrate that the conjugate can kill LHRH-R expressing triple-negative breast cancer cells (MDA-MB-231 cells) with low nanomolar IC50. Related studies on tumor-bearing mice further reveal that the same conjugate can eradicate MDA-MB-231 solid tumors without any measurable side-effects, yielding mice that gain weight during therapy and show no evidence of tumor recurrence for at least 5 weeks after termination of treatment. That these complete responses are LHRH-R targeted was then established by showing that identical treatment of receptor-negative (SKOV3) tumors yields no antitumor response. Overall, these data provide a proof-of-concept that LHRH-R specific targeting of an extremely toxic drug like tubulysin B can treat LHRH-R positive tumors without causing significant toxicity to healthy cells.