RESUMO
Cytokines are small proteins that modulate the activity of the immune system. Because of their potent immunomodulatory properties, some recombinant cytokines have undergone clinical development and have gained marketing authorization for the therapy of certain forms of cancer. Recombinant cytokines are typically administered at ultralow doses, as many of them can cause substantial toxicity even at submilligram quantities. In an attempt to increase the therapeutic index, fusion proteins based on tumor-homing antibodies (also called "immunocytokines") have been considered, and some products in this class have reached late-stage clinical trials. While antibody-cytokine fusions, which preferentially localize in the neoplastic mass, can activate tumor-resident leukocytes and may be more efficacious than their nontargeted counterparts, such products typically conserve an intact cytokine activity, which may prevent escalation to curative doses. To further improve tolerability, several strategies have been conceived for the development of antibody-cytokine fusions with "activity-on-demand", acting on tumors but helping spare normal tissues from undesired toxicity. In this article, we have reviewed some of the most promising strategies, outlining their potential as well as possible limitations.
RESUMO
DNA-Encoded Libraries (DELs) allow the parallel screening of millions of compounds for various applications, including de novo discovery or affinity maturation campaigns. However, library construction and HIT resynthesis can be cumbersome, especially when library members present an unknown stereochemistry. We introduce a permutational encoding strategy suitable for the construction of highly pure single-stranded single-pharmacophore DELs, designed to distinguish isomers at the sequencing level (e.g., stereoisomers, regio-isomers, and peptide sequences). This approach was validated by synthesizing a mock 921,600-member 4-amino-proline single-stranded DEL ("DEL1"). While screening DEL1 against different targets, high-throughput sequencing results showed selective enrichment of the most potent stereoisomers, with enrichment factors that outperform conventional encoding strategies. The versatility of our methodology was additionally validated by encoding 24 scaffolds derived from different permutations of the amino acid sequence of a previously described cyclic peptide targeting Fibroblast Activation Protein (FAP-2286). The resulting library ("DEL2") was interrogated against human FAP, showing selective enrichment of five cyclic peptides. We observed a direct correlation between enrichment factors and on-DNA binding affinities. The presented encoding methodology accelerates drug discovery by facilitating library synthesis and streamlining HIT resynthesis while enhancing enrichment factors at the DEL sequencing level. This facilitates the identification of HIT candidates prior to medicinal chemistry and affinity maturation campaigns.
Assuntos
DNA de Cadeia Simples , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Biblioteca Gênica , Descoberta de Drogas/métodos , Estereoisomerismo , Humanos , Peptídeos Cíclicos/química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Sequência de AminoácidosRESUMO
PURPOSE: Pluvicto™ ([177Lu]Lu-PSMA-617), a radioligand therapeutic targeting prostate-specific membrane antigen (PSMA), has been recently approved for the treatment of metastatic castration-resistant prostate cancer (mCRPR). The drug suffers from salivary gland and kidney uptake that prevents its dose escalation to potentially curative doses. In this work, we sought to potentiate the in vivo anti-cancer activity of Pluvicto™ by combining it with L19-IL2, a clinical-stage investigational medicinal product based on tumor-targeted interleukin-2. METHODS: We established a new PSMA-expressing model (HT-1080.hPSMA) and validated it using a fluoresceine analogue of PSMA-617 (compound 1). The HT-1080.hPSMA model was used to study the saturation and tumor retention of Pluvicto™ (compound 2) and to run combination therapy studies with L19-IL2. To complement our understanding of the mechanism of action of this novel combination, we conducted proteomics experiments on tumor samples after therapy with Pluvicto™ alone or in combination with the immunocytokine. RESULTS: High, selective, and long-lived tumor uptake was observed for Pluvicto™ (2) in the novel HT-1080.hPSMA model. Therapy studies in HT-1080.hPSMA tumor-bearing mice revealed that the combination of Pluvicto™ (2) plus L19-IL2 mediated curative and durable responses in all animals. Potent in vivo anti-cancer activity was observed solely for the combination modality, at doses that were well tolerated by treated animals. Proteomics studies indicated that L19-IL2 boosts the activation of the immune system in animals pre-treated with Pluvicto™. CONCLUSION: The therapeutic efficacy of Pluvicto™ at low radioactive doses can be effectively enhanced by the combination with L19-IL2. Our findings warrant further clinical exploration of this novel combination modality.
Assuntos
Interleucina-2 , Animais , Camundongos , Humanos , Linhagem Celular Tumoral , Masculino , Compostos Heterocíclicos com 1 Anel/química , Compostos Heterocíclicos com 1 Anel/uso terapêutico , Glutamato Carboxipeptidase II/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Antineoplásicos/farmacocinética , Dipeptídeos/uso terapêutico , Dipeptídeos/farmacologia , Dipeptídeos/farmacocinética , Lutécio/uso terapêutico , Distribuição Tecidual , Antígenos de Superfície , Antígeno Prostático EspecíficoRESUMO
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.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Endopeptidases/química , Endopeptidases/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Animais , Linhagem Celular Tumoral , Endopeptidases/fisiologia , Fibroblastos , Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/genética , Marcação por Isótopo , Ligantes , Lutécio/química , Masculino , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Nus , Neoplasias/metabolismo , Quinolinas/química , Radioisótopos/química , Compostos Radiofarmacêuticos , Distribuição Tecidual/fisiologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
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.
Assuntos
Antineoplásicos , Imunoconjugados , Neoplasias , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Fibroblastos , Oligopeptídeos , Peptídeos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Recently, Pluvicto™ ([177Lu]Lu-PSMA-617), a small-molecule prostate-specific membrane antigen (PSMA) radioligand therapeutic, has been approved by the FDA in metastatic castration-resistant prostate cancer. Pluvicto™ and other PSMA-targeting radioligand therapeutics (RLTs) have shown side effects due to accumulation in certain healthy tissues, such as salivary glands and kidney. Until now, the molecular mechanism underlying the undesired accumulation of PSMA-targeting RLTs had not been elucidated. METHODS: We compared the sequence of PSMA with the entire human proteome to identify proteins closely related to the target. We have identified glutamate carboxypeptidase III (GCPIII), N-acetylated alpha-linked acidic dipeptidase like 1 (NAALADL-1), and transferrin receptor 1 (TfR1) as extracellular targets with the highest similarity to PSMA. The affinity of compound 1 for PSMA, GCPIII, NAALADL-1, and TfR1 was measured by fluorescence polarization. The expression of the putative anti-target GCPIII was assessed by immunofluorescence on human salivary glands and kidney, using commercially available antibodies. RESULTS: A fluorescent derivative of Pluvicto™ (compound 1) bound tightly to PSMA and to GCPIII in fluorescence polarization experiments, while no interaction was observed with NAALADL-1 and TfR1. Immunofluorescence analysis revealed abundant expression of GCPIII both in healthy human kidney and salivary glands. CONCLUSION: We conclude that the membranous expression of GCPIII in kidney and salivary gland may be the underlying cause for unwanted accumulation of Pluvicto™ and other Glu-ureido PSMA radio pharmaceuticals in patients.
Assuntos
Neoplasias de Próstata Resistentes à Castração , Compostos Radiofarmacêuticos , Masculino , Humanos , Compostos Radiofarmacêuticos/uso terapêutico , Dipeptídeos/uso terapêutico , Antígeno Prostático Específico , Neoplasias de Próstata Resistentes à Castração/metabolismo , Glutamato Carboxipeptidase II/metabolismo , Antígenos de Superfície/metabolismo , Radioisótopos/uso terapêutico , Glândulas Salivares/diagnóstico por imagem , Glândulas Salivares/metabolismo , Rim/metabolismo , Lutécio/uso terapêuticoRESUMO
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.
Assuntos
Neoplasias , Compostos Radiofarmacêuticos , Humanos , Espectrometria de Massas , Metais , Radioisótopos , Reprodutibilidade dos Testes , Distribuição TecidualRESUMO
Antibody-cytokine fusion proteins (immunocytokines) are gaining importance for cancer therapy, but those products are often limited by systemic toxicity related to the activity of the cytokine payload in circulation and in secondary lymphoid organs. Tumor necrosis factor (TNF) is used as a pro-inflammatory payload to trigger haemorrhagic necrosis and boost anti-cancer immunity at the tumor site. Here we describe a depotentiated version of TNF (carrying the single point mutation I97A), which displayed reduced binding affinity to its cognate receptor tumor necrosis factor receptor 1 (TNFR-1) and lower biocidal activity. The fusion of the TNF(I97A) mutant to the L19 antibody promoted restoration of anti-tumor activity upon accumulation on the cognate antigen, the alternatively spliced EDB domain of fibronectin. In vivo administration of high doses (375 µg/Kg) of the fusion protein showed a potent anti-tumor effect without apparent toxicity compared with the wild type protein. L19-TNFI97A holds promise for the targeted delivery of TNF activity to neoplastic lesions, helping spare normal tissues.
Assuntos
Receptores do Fator de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais Humanizados/genética , Anticorpos Monoclonais Humanizados/metabolismo , Cricetulus , Citocinas/genética , Citocinas/metabolismo , Feminino , Fibronectinas/genética , Fibronectinas/metabolismo , Imunofluorescência , Imunoterapia , Camundongos Endogâmicos BALB C , Mutação , Estrutura Secundária de Proteína , Receptores do Fator de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/genéticaRESUMO
We describe the cloning and characterization of a novel fusion protein (termed L19-mIL12), consisting of murine interleukin-12 in single-chain format, sequentially fused to the L19 antibody in tandem diabody format. The fusion protein bound avidly to the cognate antigen (the alternatively spliced EDB domain of fibronectin), retained the activity of the parental cytokine and was able to selectively localize to murine tumors in vivo, as shown by quantitative biodistribution analysis. L19-mIL12 exhibited a potent antitumor activity in immunocompetent mice bearing CT26 carcinomas and WEHI-164 sarcomas, which could be boosted by combination with checkpoint blockade, leading to durable cancer eradication. L19-mIL12 also inhibited tumor growth in mice with Lewis lung carcinoma (LLC), but in this case, cancer cures could not be obtained, both in monotherapy and in combination. A microscopic analysis and a depletion experiment of tumor-infiltrating leukocytes illustrated the contribution of NK cells and CD8+ T cells for the anticancer activity observed in both tumor models. Upon L19-mIL12 treatment, the density of regulatory T cells (Tregs) was strongly increased in LLC, but not in CT26 tumors. A FACS analysis also revealed that the majority of CD8+ T cells in CT26 tumors were specific to the retroviral AH1 antigen.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Antineoplásicos Imunológicos/farmacologia , Linfócitos T CD8-Positivos/imunologia , Neoplasias do Colo/imunologia , Sinergismo Farmacológico , Interleucina-12/administração & dosagem , Células Matadoras Naturais/imunologia , Animais , Anticorpos Monoclonais/imunologia , Apoptose , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/patologia , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Carcinoma Pulmonar de Lewis/imunologia , Carcinoma Pulmonar de Lewis/patologia , Proliferação de Células , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Feminino , Fibronectinas/imunologia , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/patologia , Ativação Linfocitária , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Sarcoma/tratamento farmacológico , Sarcoma/imunologia , Sarcoma/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
All Universal Chimeric Antigen Receptor T-cells (UniCAR T-cells) are T-cells which have been engineered to recognize a haptenated ligand. Due to this feature, UniCAR T-cells have the potential to mediate a potent and selective tumor killing only in the presence of a haptenated tumor ligand, thus avoiding the long-lasting biocidal effects of conventional CAR T-cells. We have used fluorescein-labeled versions of small organic ligands and different antibody formats specific to carbonic anhydrase IX (a tumor-associated antigen) in order to assess whether the killing potential of UniCAR T-cells depended on the molecular features of the haptenated molecule. Both small molecule ligands and larger antibody fragments were potent in mediating tumor cell killing over a broad concentration range. Antibodies could be conveniently used both in IgG format and as smaller diabody fragments. Importantly, the use of site-specific chemical modification strategies for the antibody coupling to fluorescein led to a substantial improvement of tumor cell killing performance, compared to the random modification of primary amino groups on the antibody surface.
Assuntos
Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Antígenos de Neoplasias/metabolismo , Apoptose/imunologia , Anidrase Carbônica IX/metabolismo , Linhagem Celular Tumoral , Fluoresceína/química , Humanos , Cinética , Ligantes , Receptores de Antígenos Quiméricos/metabolismoRESUMO
We report a novel, fast, and automatic SPME-based method capable of extracting a small molecule-drug conjugate (SMDC) from biological matrices. Our method relies on the extraction of the drug conjugate followed by direct elution into an electrospray mass spectrometer (ESI-MS) source for qualitative and quantitative analysis. We designed a tool for extracting the targeting head of a recently synthesized SMDC, which includes acetazolamide (AAZ) as high-affinity ligand specific to carbonic anhydrase IX. Specificity of the extraction was achieved through systematic optimization. The design of the extraction tool is based on noncovalent and reversible interaction between AAZ and CAII that is immobilized on the SPME extraction phase. Using this approach, we showed a 330% rise in extracted AAZ signal intensity compared to a control, which was performed in the absence of CAII. A linear dynamic range from 1.2 to 25 µg/ml was found. The limits of detection (LOD) of extracted AAZ from phosphate-buffered saline (PBS) and human plasma were 0.4 and 1.2 µg/ml, respectively. This with a relative standard deviation of less than 14% (n = 40) covers the therapeutic range. Graphical abstract.
Assuntos
Acetazolamida/isolamento & purificação , Inibidores Enzimáticos/química , Bibliotecas de Moléculas Pequenas/isolamento & purificação , Microextração em Fase Sólida/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Acetazolamida/química , Automação , Limite de Detecção , Reprodutibilidade dos Testes , Bibliotecas de Moléculas Pequenas/químicaRESUMO
We present the first direct comparative evaluation of an antibody-drug conjugate and of a small molecule-drug conjugate for cancer therapy, using chemically defined products which bind with high-affinity to carbonic anhydrase IX, a marker of tumor hypoxia and of renal cell carcinoma.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Inibidores da Anidrase Carbônica/farmacologia , Carcinoma de Células Renais/tratamento farmacológico , Neoplasias Renais/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/farmacologia , Hipóxia Tumoral/efeitos dos fármacos , Animais , Anticorpos Monoclonais/química , Antígenos de Neoplasias/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/química , Anidrase Carbônica IX/antagonistas & inibidores , Anidrase Carbônica IX/metabolismo , Inibidores da Anidrase Carbônica/síntese química , Inibidores da Anidrase Carbônica/química , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Bibliotecas de Moléculas Pequenas/químicaAssuntos
Glutamato Carboxipeptidase II , Ureia , Humanos , Ligantes , Antígenos de Superfície , Linhagem Celular TumoralRESUMO
Antibody-drug conjugates (ADCs) represent an attractive class of biopharmaceutical agents, with the potential to selectively deliver potent cytotoxic agents to tumors. It is generally assumed that ADC products should preferably bind and internalize into cancer cells in order to liberate their toxic payload, but a growing body of evidence indicates that also ADCs based on noninternalizing antibodies may be potently active. In this Communication, we investigated dipeptide-based linkers (frequently used for internalizing ADC products) in the context of the noninternalizing F16 antibody, specific to a splice isoform of tenascin-C. Using monomethyl auristatin E (MMAE) as potent cytotoxic drug, we observed that a single amino acid substitution of the Val-Cit dipeptide linker can substantially modulate the in vivo stability of the corresponding ADC products, as well as the anticancer activity in mice bearing the human epidermoid A431 carcinoma. In these settings, the linker based on the Val-Ala dipeptide exhibited better performances, compared to Val-Cit, Val-Lys, and Val-Arg analogues. Mass spectrometric analysis revealed that the four linkers displayed not only different stability in vivo but also differences in cleavage sites. Moreover, the absence of anticancer activity for a F16-MMAE conjugate featuring a noncleavable linker indicated that drug release modalities, based on proteolytic degradation of the immunoglobulin moiety, cannot be exploited with noninternalizing antibodies. ADC products based on the noninternalizing F16 antibody may be useful for the treatment of several human malignancies, as the cognate antigen is abundantly expressed in the extracellular matrix of several tumors, while being virtually undetectable in most normal adult tissues.
Assuntos
Antineoplásicos/farmacologia , Dipeptídeos/metabolismo , Imunoconjugados/química , Peptídeo Hidrolases/metabolismo , Alanina , Aminobenzoatos/administração & dosagem , Aminobenzoatos/química , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Matriz Extracelular/química , Matriz Extracelular/imunologia , Humanos , Imunoconjugados/metabolismo , Camundongos , Oligopeptídeos/administração & dosagem , Oligopeptídeos/química , Tenascina/genética , Tenascina/imunologia , ValinaRESUMO
Cytotoxic drugs, which are commonly used for the pharmacotherapy of many forms of cancer, often cause substantial toxicity to the patient without being able to induce long-lasting remissions. Ligands specific to accessible tumor-associated targets, capable of selective localization at the neoplastic site, may facilitate the preferential delivery of anti-cancer drugs, boosting activity and helping spare normal organs. In this article, we present a critical analysis of the limitation of conventional anti-cancer drugs and we contrast monoclonal antibodies and small organic ligands, as vehicles for pharmacodelivery applications.
Assuntos
Antineoplásicos/administração & dosagem , Sistemas de Liberação de Medicamentos , Anticorpos Monoclonais/administração & dosagem , Humanos , Ligantes , Neoplasias/tratamento farmacológicoRESUMO
Fibroblast activation protein (FAP) is a protein biomarker widely expressed in most solid human malignancies of epithelial origin. In recent years, a number of FAP-targeted small organic radioligands, including OncoFAP, have been utilized in the clinic for the detection and diagnosis of cancer. Despite their selective accumulation, conventional FAP ligands present a relatively short half-life in tumors, corresponding to a few hours after systemic administration. In order to maximize their efficacy, FAP-targeted radioligand therapeutics must possess prolonged tumor retention, thus irradiating tumor cells for days. In this work, we describe the development of compact OncoFAP multimers with improved FAP affinity (low picomolar IC50s), aimed at increasing tumor-residence time for therapeutic applications. An in silico analysis of the interaction of the multimers with FAP revealed a wide and deep pocket and six additional secondary binding sites. TriOncoFAP-DOTAGA emerged for its favorable in vitro profile and superior in vivo biodistribution performance in tumor-bearing mice.
Assuntos
Endopeptidases , Animais , Humanos , Camundongos , Endopeptidases/metabolismo , Distribuição Tecidual , Proteínas de Membrana/metabolismo , Linhagem Celular Tumoral , Serina Endopeptidases/metabolismo , Gelatinases/metabolismo , Feminino , Neoplasias/tratamento farmacológicoRESUMO
Prostate-specific membrane antigen (PSMA)-targeted radio ligand therapeutics (RLTs), such as [177Lu]Lu-PSMA-617 (Pluvicto), have been shown to accumulate in salivary glands and kidneys, potentially leading to undesired side effects. As unwanted accumulation in normal organs may derive from the cross-reactivity of PSMA ligands to glutamate carboxypeptidase III (GCPIII), it may be convenient to block this interaction with GCPIII-selective ligands. Parallel screening of a DNA-encoded chemical library (DEL) against GCPIII and PSMA allowed the identification of GCPIII binders. Structure-activity relationship (SAR) studies resulted in the identification of nanomolar GCPIII ligands with up to 1000-fold selectivity over PSMA. We studied the ability of GCPIII ligands to counteract the binding of [177Lu]Lu-PSMA-617 to human salivary glands by autoradiography and could demonstrate a partial radioprotection.
Assuntos
Dipeptídeos , Compostos Heterocíclicos com 1 Anel , Lutécio , Humanos , Antígenos de Superfície , Autorradiografia , Dipeptídeos/química , Dipeptídeos/metabolismo , Glutamato Carboxipeptidase II , Compostos Heterocíclicos com 1 Anel/química , Compostos Heterocíclicos com 1 Anel/metabolismo , Ligantes , Lutécio/química , Lutécio/metabolismo , Antígeno Prostático Específico , Radioisótopos/química , Radioisótopos/metabolismo , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/metabolismo , Compostos Radiofarmacêuticos/farmacocinética , Glândulas Salivares/metabolismo , Relação Estrutura-Atividade , Distribuição TecidualRESUMO
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.
RESUMO
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.
Assuntos
Antineoplásicos , Imunoconjugados , Humanos , Animais , Camundongos , Preparações Farmacêuticas , Tubulina (Proteína) , Microambiente Tumoral , Imunoconjugados/uso terapêutico , Imunoconjugados/química , Camptotecina/uso terapêutico , Linhagem Celular TumoralRESUMO
DNA-encoded chemical libraries (DELs) are powerful drug discovery tools, enabling the parallel screening of millions of DNA-barcoded compounds. We investigated how the DEL input affects the hit discovery rate in DEL screenings. Evaluation of selection fingerprints revealed that the use of approximately 105 copies of each library member is required for the confident identification of nanomolar hits, using generally applicable methodologies.