DNA-encoded chemical libraries enable the discovery of potent PSMA-ligands with substantially reduced affinity towards the GCPIII anti-target.

Prostate-specific membrane antigen (PSMA) is a tumor-associated protein that has been successfully targeted with small organic ligands and monoclonal antibodies. Pluvicto™ is a PSMA-targeted radioligand therapeutic (RLT) recently approved by the FDA for the treatment of metastatic castration-resistant prostate cancer (2022 FDA marketing authorization). Although a large Phase III clinical trial (VISION trial) demonstrated clinical benefits in patients treated with Pluvicto™, the therapeutic window of the drug is narrowed by its undesired accumulation in healthy organs. Glutamate carboxypeptidase III (GCPIII), an enzyme sharing 70% identity with PSMA, may be responsible for the off-target accumulation of PSMA-RLTs in salivary glands and kidneys. In this work, we designed and synthesized affinity and selectivity maturation DNA-encoded chemical libraries (ASM-DELs) comprising 18'284'658 compounds that were screened in parallel against PSMA and GCPIII with the aim to identify potent and selective PSMA ligands for tumor-targeting applications. Compound A70-B104 was isolated as the most potent and selective ligand (KD of 900 pM for PSMA, KD of 40 nM for GCPIII). 177Lu-A70-B104-DOTA, a radiolabeled derivative of compound A70-B104, presented selective accumulation in PSMA-positive cancer lesions (i.e., 7.4% ID g-1, 2 hour time point) after systemic administration in tumor-bearing mice. The results of autoradiography experiments showed that 177Lu-A70-B104-DOTA selectively binds to PSMA-positive cancer tissues, while negligible binding on human salivary glands was observed.

In vivo activation of FAP-cleavable small molecule-drug conjugates for the targeted delivery of camptothecins and tubulin poisons to the tumor microenvironment.

Small molecule-drug conjugates (SMDCs) are increasingly considered as a therapeutic alternative to antibody-drug conjugates (ADCs) for cancer therapy. OncoFAP is an ultra-high affinity ligand of Fibroblast Activation Protein (FAP), a stromal tumor-associated antigen overexpressed in a wide variety of solid human malignancies. We have recently reported the development of non-internalizing OncoFAP-based SMDCs, which are activated by FAP thanks to selective proteolytic cleavage of the -GlyPro- linker with consequent release of monomethyl auristatin E (MMAE) in the tumor microenvironment. In this article, we describe the generation and the in vivo characterization of FAP-cleavable OncoFAP-drug conjugates based on potent topoisomerase I inhibitors (DXd, SN-38, and exatecan) and an anti-tubulin payload (MMAE), which are already exploited in clinical-stage and approved ADCs. The Glycine-Proline FAP-cleavable technology was directly benchmarked against linkers found in Adcetris™, Enhertu™, and Trodelvy™ structures by means of in vivo therapeutic experiments in mice bearing tumors with cellular or stromal FAP expression. OncoFAP-GlyPro-Exatecan and OncoFAP-GlyPro-MMAE emerged as the most efficacious anti-cancer therapeutics against FAP-positive cellular models. OncoFAP-GlyPro-MMAE exhibited a potent antitumor activity also against stromal models, and was therefore selected for clinical development.

Ex vivo mass spectrometry-based biodistribution analysis of an antibody-Resiquimod conjugate bearing a protease-cleavable and acid-labile linker.

Immune-stimulating antibody conjugates (ISACs) equipped with imidazoquinoline (IMD) payloads can stimulate endogenous immune cells to kill cancer cells, ultimately inducing long-lasting anticancer effects. A novel ISAC was designed, featuring the IMD Resiquimod (R848), a tumor-targeting antibody specific for Carbonic Anhydrase IX (CAIX) and the protease-cleavable Val-Cit-PABC linker. In vitro stability analysis showed not only R848 release in the presence of the protease Cathepsin B but also under acidic conditions. The ex vivo mass spectrometry-based biodistribution data confirmed the low stability of the linker-drug connection while highlighting the selective accumulation of the IgG in tumors and its long circulatory half-life.

Tumor-Targeted Interleukin 2 Boosts the Anticancer Activity of FAP-Directed Radioligand Therapeutics.

We studied the antitumor efficacy of a combination of 177Lu-labeled radioligand therapeutics targeting the fibroblast activation protein (FAP) (OncoFAP and BiOncoFAP) with the antibody-cytokine fusion protein L19-interleukin 2 (L19-IL2) providing targeted delivery of interleukin 2 to tumors. Methods: The biodistribution of 177Lu-OncoFAP and 177Lu-BiOncoFAP at different molar amounts (3 vs. 250 nmol/kg) of injected ligand was studied via SPECT/CT in mice bearing subcutaneous HT-1080.hFAP tumors, and self-absorbed tumor and organ doses were calculated. The in vivo anticancer effect of 5 MBq of the radiolabeled preparations was evaluated as monotherapy or in combination with L19-IL2 in subcutaneously implanted HT-1080.hFAP and SK-RC-52.hFAP tumors. Tumor samples from animals treated with 177Lu-BiOncoFAP, L19-IL2, or both were analyzed by mass spectrometry-based proteomics to identify therapeutic signatures on cellular and stromal markers of cancer and on immunomodulatory targets. Results: 177Lu-BiOncoFAP led to a significantly higher self-absorbed dose in FAP-positive tumors (0.293 ± 0.123 Gy/MBq) than did 177Lu-OncoFAP (0.157 ± 0.047 Gy/MBq, P = 0.01) and demonstrated favorable tumor-to-organ ratios at high molar amounts of injected ligand. Administration of L19-IL2 or 177Lu-BiOncoFAP as single agents led to cancer cures in only a limited number of treated animals. In 177Lu-BiOncoFAP-plus-L19-IL2 combination therapy, complete remissions were observed in all injected mice (7/7 complete remissions for the HT-1080.hFAP model, and 4/4 complete remissions for the SK-RC-52.hFAP model), suggesting therapeutic synergy. Proteomic studies revealed a mechanism of action based on the activation of natural killer cells, with a significant enhancement of the expression of granzymes and perforin 1 in the tumor microenvironment after combination treatment. Conclusion: The combination of OncoFAP-based radioligand therapeutics with concurrent targeting of interleukin 2 shows synergistic anticancer effects in the treatment of FAP-positive tumors. This experimental finding should be corroborated by future clinical studies.

A Comparative Analysis of Fibroblast Activation Protein-Targeted Small Molecule-Drug, Antibody-Drug, and Peptide-Drug Conjugates.

We present the first in vivo comparative evaluation of chemically defined antibody-drug conjugates (ADCs), small molecule-drug conjugates (SMDCs), and peptide-drug conjugates (PDCs) targeting and activated by fibroblast activation protein (FAP) in solid tumors. Both the SMDC (OncoFAP-Gly-Pro-MMAE) and the ADC (7NP2-Gly-Pro-MMAE) candidates delivered high amounts of active payload (i.e., MMAE) selectively at the tumor site, thus producing a potent antitumor activity in a preclinical cancer model.

Fibroblast Activation Protein Triggers Release of Drug Payload from Non-internalizing Small Molecule Drug Conjugates in Solid Tumors.

PURPOSE: Small molecule drug conjugates (SMDC) are modular anticancer prodrugs that include a tumor-targeting small organic ligand, a cleavable linker, and a potent cytotoxic agent. Most of the SMDC products that have been developed for clinical applications target internalizing tumor-associated antigens on the surface of tumor cells. We have recently described a novel non-internalizing small organic ligand (named OncoFAP) of fibroblast activation protein (FAP), a tumor-associated antigen highly expressed in the stroma of most solid human malignancies. EXPERIMENTAL DESIGN: In this article, we describe a new series of OncoFAP-Drug derivatives based on monomethyl auristatin E (MMAE; a potent cytotoxic tubulin poison) and dipeptide linkers that are selectively cleaved by FAP in the tumor microenvironment. RESULTS: The tumor-targeting potential of OncoFAP was confirmed in patients with cancer using nuclear medicine procedures. We used mass spectrometry methodologies to quantify the amount of prodrug delivered to tumors and normal organs, as well as the efficiency of the drug release process. Linkers previously exploited for anticancer drug conjugates were used as benchmark. We identified OncoFAP-Gly-Pro-MMAE as the best performing SMDC, which has now been prioritized for further clinical development. OncoFAP-Gly-Pro-MMAE selectively delivered more than 10% injected dose per gram of MMAE to FAP-positive tumors, with a tumor-to-kidney ratio of 16:1 at 24 hours post-injection. CONCLUSIONS: The FAP-specific drug conjugates described in this article promise to be efficacious for the targeting of human malignancies. The extracellular release of potent anticancer payloads mediates durable complete remission in difficult-to-treat animal models of cancer.

A Dimeric FAP-Targeting Small-Molecule Radioconjugate with High and Prolonged Tumor Uptake.

Imaging procedures based on small-molecule radioconjugates targeting fibroblast activation protein (FAP) have recently emerged as a powerful tool for the diagnosis of a wide variety of tumors. However, the therapeutic potential of radiolabeled FAP-targeting agents is limited by their short residence time in neoplastic lesions. In this work, we present the development and in vivo characterization of BiOncoFAP, a new dimeric FAP-binding motif with an extended tumor residence time and favorable tumor-to-organ ratio. Methods: The binding properties of BiOncoFAP and its monovalent OncoFAP analog were assayed against recombinant human FAP. Preclinical experiments with 177Lu-OncoFAP-DOTAGA (177Lu-OncoFAP) and 177Lu-BiOncoFAP-DOTAGA (177Lu-BiOncoFAP) were performed on mice bearing FAP-positive HT-1080 tumors. Results: OncoFAP and BiOncoFAP displayed comparable subnanomolar dissociation constants toward recombinant human FAP in solution, but the bivalent BiOncoFAP bound more avidly to the target immobilized on solid supports. In a comparative biodistribution study, 177Lu-BiOncoFAP exhibited a more stable and prolonged tumor uptake than 177Lu-OncoFAP (∼20 vs. ∼4 percentage injected dose/g, respectively, at 24 h after injection). Notably, 177Lu-BiOncoFAP showed favorable tumor-to-organ ratios with low kidney uptake. Both 177Lu-OncoFAP and 177Lu-BiOncoFAP displayed potent antitumor efficacy when administered at therapeutic doses to tumor-bearing mice. Conclusion: 177Lu-BiOncoFAP is a promising candidate for radioligand therapy of cancer, with favorable in vivo tumor-to-organ ratios, a long tumor residence time, and potent anticancer efficacy.