An ultra-high-affinity small organic ligand of fibroblast activation protein for tumor-targeting applications.

We describe the development of OncoFAP, an ultra-high-affinity ligand of fibroblast activation protein (FAP) for targeting applications with pan-tumoral potential. OncoFAP binds to human FAP with affinity in the subnanomolar concentration range and cross-reacts with the murine isoform of the protein. We generated various fluorescent and radiolabeled derivatives of OncoFAP in order to study biodistribution properties and tumor-targeting performance in preclinical models. Fluorescent derivatives selectively localized in FAP-positive tumors implanted in nude mice with a rapid and homogeneous penetration within the neoplastic tissue. Quantitative in vivo biodistribution studies with a lutetium-177-labeled derivative of OncoFAP revealed a preferential localization in tumors at doses of up to 1,000 nmol/kg. More than 30% of the injected dose had already accumulated in 1 g of tumor 10 min after intravenous injection and persisted for at least 3 h with excellent tumor-to-organ ratios. OncoFAP also served as a modular component for the generation of nonradioactive therapeutic products. A fluorescein conjugate mediated a potent and FAP-dependent tumor cell killing activity in combination with chimeric antigen receptor (CAR) T cells specific to fluorescein. Similarly, a conjugate of OncoFAP with the monomethyl auristatin E-based Vedotin payload was well tolerated and cured tumor-bearing mice in combination with a clinical-stage antibody-interleukin-2 fusion. Collectively, these data support the development of OncoFAP-based products for tumor-targeting applications in patients with cancer.

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.

Role of Stereochemistry on the Biological Activity of Nature-Inspired 3-Br-Acivicin Isomers and Derivatives.

Chiral natural compounds are often biosynthesized in an enantiomerically pure fashion, and stereochemistry plays a pivotal role in biological activity. Herein, we investigated the significance of chirality for nature-inspired 3-Br-acivicin (3-BA) and its derivatives. The three unnatural isomers of 3-BA and its ester and amide derivatives were prepared and characterized for their antimalarial activity. Only the (5S, αS) isomers displayed significant antiplasmodial activity, revealing that their uptake might be mediated by the L-amino acid transport system, which is known to mediate the acivicin membrane's permeability. In addition, we investigated the inhibitory activity towards Plasmodium falciparum glyceraldehyde 3-phosphate dehydrogenase (PfGAPDH) since it is involved in the multitarget mechanism of action of 3-BA. Molecular modeling has shed light on the structural and stereochemical requirements for an efficient interaction with PfGAPDH, leading to covalent irreversible binding and enzyme inactivation. While stereochemistry affects the target binding only for two subclasses (1a-d and 4a-d), it leads to significant differences in the antimalarial activity for all subclasses, suggesting that a stereoselective uptake might be responsible for the enhanced biological activity of the (5S, αS) isomers.

Mass Spectrometry-Based Method for the Determination of the Biodistribution of Tumor-Targeting Small Molecule-Metal Conjugates.

Nuclear medicine plays a key role in modern diagnosis and cancer therapy. The development of tumor-targeting radionuclide conjugates (also named small molecule-radio conjugates (SMRCs)) represents a significant improvement over the clinical use of metabolic radiotracers (e.g., [18F]-fluorodeoxyglucose) for imaging and over the application of biocidal external beam radiations for therapy. During the discovery of SMRCs, molecular candidates must be carefully evaluated typically by performing biodistribution assays in preclinical tumor models. Quantification methodologies based on radioactive counts are typically demanding due to safety concerns, availability of radioactive materials, and infrastructures. In this article, we report the development of a mass spectrometry (MS)-based method for the detection and quantification of small molecule-metal conjugates (SMMCs) as cold surrogates of SMRCs. We applied this methodology for the evaluation of the biodistribution of a particular class of tumor-targeting drug candidates based on natLu, natGa, and natF and directed against fibroblast activation protein (FAP). The reliability of the liquid chromatography-MS (LC-MS) analysis was validated by a direct comparison of MS-based and radioactivity-based biodistribution data. The results show that MS biodistribution of stable isotope metal conjugates is an orthogonal tool for the preclinical characterization of different classes of radiopharmaceuticals.

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.

Discovery of a spirocyclic 3-bromo-4,5-dihydroisoxazole covalent inhibitor of hGAPDH with antiproliferative activity against pancreatic cancer cells.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key glycolytic enzyme, plays a crucial role in the energy metabolism of cancer cells and has been proposed as a valuable target for the development of anticancer agents. Among a series of 5-substituted 3-bromo-4,5-dihydroisoxazole (BDHI) derivatives, we identified the spirocyclic compound 11, which is able to covalently inactivate recombinant human GAPDH (hGAPDH) with a faster reactivity than koningic acid, one of the most potent hGAPDH inhibitors known to date. Computational studies confirmed that conformational rigidification is crucial to stabilize the interaction of the inhibitor with the binding site, thus favoring the subsequent covalent bond formation. Investigation of intrinsic warhead reactivity at different pH disclosed the negligible reactivity of 11 with free thiols, highlighting its ability to selectively react with the activated cysteine of hGAPDH with respect to other sulfhydryl groups. Compound 11 strongly reduced cancer cell growth in four different pancreatic cancer cell lines and its antiproliferative activity correlated well with the intracellular inhibition of hGAPDH. Overall, our results qualify 11 as a potent hGAPDH covalent inhibitor with a moderate drug-like reactivity that could be further exploited to develop anticancer agents.

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.

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.

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.

Automated Radiosynthesis, Preliminary In Vitro/In Vivo Characterization of OncoFAP-Based Radiopharmaceuticals for Cancer Imaging and Therapy.

FAP-targeted radiopharmaceuticals represent a breakthrough in cancer imaging and a viable option for therapeutic applications. OncoFAP is an ultra-high-affinity ligand of FAP with a dissociation constant of 680 pM. OncoFAP has been recently discovered and clinically validated for PET imaging procedures in patients with solid malignancies. While more and more clinical validation is becoming available, the need for scalable and robust procedures for the preparation of this new class of radiopharmaceuticals continues to increase. In this article, we present the development of automated radiolabeling procedures for the preparation of OncoFAP-based radiopharmaceuticals for cancer imaging and therapy. A new series of [68Ga]Ga-OncoFAP, [177Lu]Lu-OncoFAP and [18F]AlF-OncoFAP was produced with high radiochemical yields. Chemical and biochemical characterization after radiolabeling confirmed its excellent stability, retention of high affinity for FAP and absence of radiolysis by-products. The in vivo biodistribution of [18F]AlF-NOTA-OncoFAP, a candidate for PET imaging procedures in patients, was assessed in mice bearing FAP-positive solid tumors. The product showed rapid accumulation in solid tumors, with an average of 6.6% ID/g one hour after systemic administration and excellent tumor-to-healthy organs ratio. We have developed simple, quick, safe and robust synthetic procedures for the preparation of theranostic OncoFAP-compounds based on Gallium-68, Lutetium-177 and Fluorine-18 using the commercially available FASTlab synthesis module.

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.

Development of Potent 3-Br-isoxazoline-Based Antimalarial and Antileishmanial Compounds.

Starting from the structure of previously reported 3-Br-isoxazoline-based covalent inhibitors of P. falciparum glyceraldehyde 3-phosphate dehydrogenase, and with the intent to improve their metabolic stability and antimalarial activity, we designed and synthesized a series of simplified analogues that are characterized by the insertion of the oxadiazole ring as a bioisosteric replacement for the metabolically labile ester/amide function. We then further replaced the oxadiazole ring with a series of five-membered heterocycles and finally combined the most promising structural features. All the new derivatives were tested in vitro for antimalarial as well as antileishmanial activity. We identified two very promising new lead compounds, endowed with submicromolar antileishmanial activity and nanomolar antiplasmodial activity, respectively, and a very high selectivity index with respect to mammalian cells.

Immunotherapy with Immunocytokines and PD-1 Blockade Enhances the Anticancer Activity of Small Molecule-Drug Conjugates Targeting Carbonic Anhydrase IX.

Small molecule-drug conjugates (SMDCs) represent an alternative to conventional antitumor chemotherapeutic agents, with the potential to improve the therapeutic window of cytotoxic payloads through active delivery at the site of the disease. In this article, we describe novel combination therapies consisting of anti-carbonic anhydrase IX SMDCs combined with different immunomodulatory products. The therapeutic effect of the SMDCs was potentiated by combination with PD-1 blockade and with tumor-homing antibody-cytokine fusions in mouse models of renal cell carcinoma and colorectal cancer. The combination with L19-IL12, a fusion protein specific to the alternatively spliced EDB domain of fibronectin containing the murine IL12 moiety, was also active against large established tumors. Analysis of the microscopic structures of healthy organs performed 3 months after tumor eradication confirmed absence of pathologic abnormalities in the healthy kidney, liver, lung, stomach, and intestine. Our findings may be of clinical significance as they provide motivation for the development of combinations based on SMDCs and immunotherapy for the treatment of renal cell carcinoma and hypoxic tumors.

Covalent inhibitors of GAPDH: From unspecific warheads to selective compounds.

Targeting glycolysis is an attractive approach for the treatment of a wide range of pathologies, such as various tumors and parasitic infections. Due to its pivotal role in the glycolysis, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) represents a rate-limiting enzyme in those cells that mostly, or exclusively rely on this pathway for energy production. In this context, GAPDH inhibition can be a valuable approach for the development of anticancer and antiparasitic drugs. In addition to its glycolytic role, GAPDH possesses several moonlight functions, whose deregulation is involved in some pathological conditions. Covalent modification on different amino acids of GAPDH, in particular on cysteine residues, can lead to a modulation of the enzyme activity. The selectivity towards specific cysteine residues is essential to achieve a specific phenotypic effect. In this work we report an extensive overview of the latest advances on the numerous compounds able to inhibit GAPDH through the covalent binding to cysteine residues, ranging from endogenous metabolites and xenobiotics, which may serve as pharmacological tools to actual drug-like compounds with promising therapeutic perspectives. Furthermore, we focused on the potentialities of the different warheads, shedding light on the possibility to exploit a combination of a finely tuned electrophilic group with a well-designed recognition moiety. These findings can provide useful information for the rational design of novel covalent inhibitors of GAPDH, with the final goal to expand the current treatment options.