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1.
Annu Rev Biochem ; 87: 479-502, 2018 06 20.
Artículo en Inglés | MEDLINE | ID: mdl-29328784

RESUMEN

The discovery of organic ligands that bind specifically to proteins is a central problem in chemistry, biology, and the biomedical sciences. The encoding of individual organic molecules with distinctive DNA tags, serving as amplifiable identification bar codes, allows the construction and screening of combinatorial libraries of unprecedented size, thus facilitating the discovery of ligands to many different protein targets. Fundamentally, one links powers of genetics and chemical synthesis. After the initial description of DNA-encoded chemical libraries in 1992, several experimental embodiments of the technology have been reduced to practice. This review provides a historical account of important milestones in the development of DNA-encoded chemical libraries, a survey of relevant ongoing research activities, and a glimpse into the future.


Asunto(s)
Descubrimiento de Drogas/métodos , Biblioteca de Genes , Bibliotecas de Moléculas Pequeñas , Animales , Técnicas Químicas Combinatorias , Humanos , Ligandos , Biblioteca de Péptidos
2.
Mol Cell ; 80(3): 423-436.e9, 2020 11 05.
Artículo en Inglés | MEDLINE | ID: mdl-33022275

RESUMEN

The ubiquitin system regulates the DNA damage response (DDR) by modifying histone H2A at Lys15 (H2AK15ub) and triggering downstream signaling events. Here, we find that phosphorylation of ubiquitin at Thr12 (pUbT12) controls the DDR by inhibiting the function of 53BP1, a key factor for DNA double-strand break repair by non-homologous end joining (NHEJ). Detectable as a chromatin modification on H2AK15ub, pUbT12 accumulates in nuclear foci and is increased upon DNA damage. Mutating Thr12 prevents the removal of ubiquitin from H2AK15ub by USP51 deubiquitinating enzyme, leading to a pronounced accumulation of ubiquitinated chromatin. Chromatin modified by pUbT12 is inaccessible to 53BP1 but permissive to the homologous recombination (HR) proteins RNF169, RAD51, and the BRCA1/BARD1 complex. Phosphorylation of ubiquitin at Thr12 in the chromatin context is a new histone mark, H2AK15pUbT12, that regulates the DDR by hampering the activity of 53BP1 at damaged chromosomes.


Asunto(s)
Daño del ADN/fisiología , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Ubiquitina/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Cromatina/metabolismo , ADN/metabolismo , Roturas del ADN de Doble Cadena , Daño del ADN/genética , Reparación del ADN por Unión de Extremidades/genética , Reparación del ADN/genética , Proteínas de Unión al ADN/metabolismo , Histonas/metabolismo , Recombinación Homóloga/fisiología , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Fosforilación , Transducción de Señal/genética , Treonina/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/fisiología , Ubiquitina/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación
3.
Proc Natl Acad Sci U S A ; 120(34): e2304071120, 2023 08 22.
Artículo en Inglés | MEDLINE | ID: mdl-37585458

RESUMEN

Class IA phosphoinositide 3-kinase alpha (PI3Kα) is an important drug target because it is one of the most frequently mutated proteins in human cancers. However, small molecule inhibitors currently on the market or under development have safety concerns due to a lack of selectivity. Therefore, other chemical scaffolds or unique mechanisms of catalytic kinase inhibition are needed. Here, we report the cryo-electron microscopy structures of wild-type PI3Kα, the dimer of p110α and p85α, in complex with three Y-shaped ligands [cpd16 (compound 16), cpd17 (compound 17), and cpd18 (compound 18)] of different affinities and no inhibitory effect on the kinase activity. Unlike ATP-competitive inhibitors, cpd17 adopts a Y-shaped conformation with one arm inserted into a binding pocket formed by R770 and W780 and the other arm lodged in the ATP-binding pocket at an angle that is different from that of the ATP phosphate tail. Such a special interaction induces a conformation of PI3Kα resembling that of the unliganded protein. These observations were confirmed with two isomers (cpd16 and cpd18). Further analysis of these Y-shaped ligands revealed the structural basis of differential binding affinities caused by stereo- or regiochemical modifications. Our results may offer a different direction toward the design of therapeutic agents against PI3Kα.


Asunto(s)
Fosfatidilinositol 3-Quinasa , Fosfatidilinositol 3-Quinasas , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Ligandos , Microscopía por Crioelectrón , Adenosina Trifosfato/metabolismo
4.
Bioconjug Chem ; 2024 Jun 17.
Artículo en Inglés | MEDLINE | ID: mdl-38885090

RESUMEN

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.

5.
Bioconjug Chem ; 35(7): 1033-1043, 2024 Jul 17.
Artículo en Inglés | MEDLINE | ID: mdl-38963407

RESUMEN

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.


Asunto(s)
ADN de Cadena Simple , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , Biblioteca de Genes , Descubrimiento de Drogas/métodos , Estereoisomerismo , Humanos , Péptidos Cíclicos/química , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Secuencia de Aminoácidos
6.
Eur J Nucl Med Mol Imaging ; 51(8): 2332-2337, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-38563883

RESUMEN

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.


Asunto(s)
Interleucina-2 , Animales , Ratones , Humanos , Línea Celular Tumoral , Masculino , Compuestos Heterocíclicos con 1 Anillo/química , Compuestos Heterocíclicos con 1 Anillo/uso terapéutico , Glutamato Carboxipeptidasa II/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Antineoplásicos/farmacocinética , Dipéptidos/uso terapéutico , Dipéptidos/farmacología , Dipéptidos/farmacocinética , Lutecio/uso terapéutico , Distribución Tisular , Antígenos de Superficie , Antígeno Prostático Específico
7.
Proc Natl Acad Sci U S A ; 118(16)2021 04 20.
Artículo en Inglés | MEDLINE | ID: mdl-33850024

RESUMEN

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.


Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Endopeptidasas/química , Endopeptidasas/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Animales , Línea Celular Tumoral , Endopeptidasas/fisiología , Fibroblastos , Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/genética , Marcaje Isotópico , Ligandos , Lutecio/química , Masculino , Proteínas de la Membrana/fisiología , Ratones , Ratones Desnudos , Neoplasias/metabolismo , Quinolinas/química , Radioisótopos/química , Radiofármacos , Distribución Tisular/fisiología , Ensayos Antitumor por Modelo de Xenoinjerto/métodos
8.
Bioconjug Chem ; 34(7): 1205-1211, 2023 07 19.
Artículo en Inglés | MEDLINE | ID: mdl-37399501

RESUMEN

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.


Asunto(s)
Antineoplásicos , Inmunoconjugados , Neoplasias , Humanos , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Fibroblastos , Oligopéptidos , Péptidos , Ensayos Antitumor por Modelo de Xenoinjerto
9.
Eur J Clin Invest ; 53(3): e13907, 2023 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-36377348

RESUMEN

AIMS: Pulmonary hypertension (PH) is accompanied by pulmonary vascular remodelling. By targeted delivery of Interleukin-9 (IL9) via the immunocytokine F8IL9, beneficial effects could be demonstrated in a mouse model of PH. This study aimed to compare two immunocytokine formats (single-chain Fv and full IgG) and to identify potential target cells of IL9. METHODS: The Monocrotaline mouse model of PH (PH, n = 12) was chosen to evaluate the treatment effects of F8IL9F8 (n = 12) and F8IgGIL9 (n = 6) compared with sham-induced animals (control, n = 10), the dual endothelin receptor antagonist Macitentan (MAC, n = 12) or IL9-based immunocytokines with irrelevant antigen specificity (KSFIL9KSF, n = 12; KSFIgGIL9 n = 6). Besides comparative validation of treatment effects, the study was focused on the detection and quantification of mast cells (MCs) and regulatory T cells (Tregs). RESULTS: There was a significantly elevated systolic right ventricular pressure (104 ± 36 vs. 45 ± 17 mmHg) and an impairment of right ventricular echocardiographic parameters (RVbasal: 2.52 ± 0.25 vs. 1.94 ± 0.13 mm) in untreated PH compared with controls (p < 0.05). Only the groups treated with F8IL9, irrespective of the format, showed consistent beneficial effects (p < 0.05). Moreover, F8IL9F8 but not F8IgGIL9 treatment significantly reduced lung tissue damage compared with untreated PH mice (p < 0.05). There was a significant increase in Tregs in F8IL9-treated compared with control animals, the untreated PH and the MAC group (p < 0.05). CONCLUSIONS: Beneficial treatment effects of targeted IL9 delivery in a preclinical model of PH could be convincingly validated. IL9-mediated recruitment of Tregs into lung tissue might play a crucial role in the induction of anti-inflammatory and anti-proliferative mechanisms potentially contributing to a novel disease-modifying concept.


Asunto(s)
Hipertensión Pulmonar , Ratones , Animales , Hipertensión Pulmonar/tratamiento farmacológico , Interleucina-9/efectos adversos , Pulmón , Modelos Animales de Enfermedad
10.
Eur J Nucl Med Mol Imaging ; 50(3): 957-961, 2023 02.
Artículo en Inglés | MEDLINE | ID: mdl-36184692

RESUMEN

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.


Asunto(s)
Neoplasias de la Próstata Resistentes a la Castración , Radiofármacos , Masculino , Humanos , Radiofármacos/uso terapéutico , Dipéptidos/uso terapéutico , Antígeno Prostático Específico , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Glutamato Carboxipeptidasa II/metabolismo , Antígenos de Superficie/metabolismo , Radioisótopos/uso terapéutico , Glándulas Salivales/diagnóstico por imagen , Glándulas Salivales/metabolismo , Riñón/metabolismo , Lutecio/uso terapéutico
11.
Proc Natl Acad Sci U S A ; 117(50): 31780-31788, 2020 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-33239441

RESUMEN

Engineered cytokines are gaining importance in cancer therapy, but these products are often limited by toxicity, especially at early time points after intravenous administration. 4-1BB is a member of the tumor necrosis factor receptor superfamily, which has been considered as a target for therapeutic strategies with agonistic antibodies or using its cognate cytokine ligand, 4-1BBL. Here we describe the engineering of an antibody fusion protein, termed F8-4-1BBL, that does not exhibit cytokine activity in solution but regains biological activity on antigen binding. F8-4-1BBL bound specifically to its cognate antigen, the alternatively spliced EDA domain of fibronectin, and selectively localized to tumors in vivo, as evidenced by quantitative biodistribution experiments. The product promoted a potent antitumor activity in various mouse models of cancer without apparent toxicity at the doses used. F8-4-1BBL represents a prototype for antibody-cytokine fusion proteins, which conditionally display "activity on demand" properties at the site of disease on antigen binding and reduce toxicity to normal tissues.


Asunto(s)
Ligando 4-1BB/administración & dosificación , Antígenos de Neoplasias/administración & dosificación , Neoplasias/tratamiento farmacológico , Proteínas Recombinantes de Fusión/administración & dosificación , Ligando 4-1BB/genética , Ligando 4-1BB/inmunología , Animales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Línea Celular Tumoral/trasplante , Modelos Animales de Enfermedad , Femenino , Fibronectinas/genética , Fibronectinas/inmunología , Humanos , Ratones , Neoplasias/inmunología , Dominios Proteicos/genética , Dominios Proteicos/inmunología , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología
12.
Anal Chem ; 94(30): 10715-10721, 2022 08 02.
Artículo en Inglés | MEDLINE | ID: mdl-35820828

RESUMEN

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.


Asunto(s)
Neoplasias , Radiofármacos , Humanos , Espectrometría de Masas , Metales , Radioisótopos , Reproducibilidad de los Resultados , Distribución Tisular
13.
J Struct Biol ; 213(1): 107696, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33493635

RESUMEN

The use of immunomodulatory agents for the treatment of cancer is gaining a growing biopharmaceutical interest. Antibody-cytokine fusion proteins, namely immunocytokines, represent a promising solution for the regulation of the immune system at the site of disease. The three-dimensional arrangement of these molecules can profoundly influence their biological activity and pharmacokinetic properties. Structural techniques might provide important insight in the 3D arrangement of immunocytokines. Here, we performed structure investigations on clinical grade fusion proteins L19-IL2, IL12-L19L19 and L19L19-IL2 to elucidate their quaternary organization. Crystallographic characterization of the common L19 antibody fragment at a resolution of 2.0-Å was combined with low-resolution studies of the full-length chimeric molecules using small-angle synchrotron X-ray scattering (SAXS) and negative stain electron microscopy. Characterization of the full-length quaternary structures of the immunocytokines in solution by SAXS consistently supported the diabody structure in the L19-IL2 immunocytokine and allowed generation of low-resolution models of the chimeric proteins L19L19-IL2 and IL12-L19L19. Comparison with 3D reconstructions obtained from negative-stain electron microscopy revealed marked flexibility associated to the linker regions connecting the cytokine and the antibody components of the chimeric proteins. Collectively, our results indicate that low-resolution molecular structure characterizations provide useful complementary insights for the quality control of immunocytokines, constituting a powerful tool to guide the design and the subsequent optimization steps towards clinical enhancement of these chimeric protein reagents.


Asunto(s)
Citocinas/química , Animales , Línea Celular Tumoral , Humanos , Ratones , Estructura Molecular , Proteínas Recombinantes de Fusión/química , Dispersión del Ángulo Pequeño , Difracción de Rayos X/métodos
14.
Eur J Immunol ; 50(10): 1591-1597, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32470143

RESUMEN

Mice bearing CT26 tumors can be cured by administration of L19-mIL12 or F8-mTNF, two antibody fusion proteins which selectively deliver their cytokine payload to the tumor. In both settings, cancer cures crucially depended on CD8+ T cells and the AH1 peptide (derived from the gp70 protein of the murine leukemia virus) acted as the main tumor-rejection antigen, with ∼50% of CD8+ T cells in the neoplastic mass being AH1-specific after therapy. In order to characterize the clonality of the T cell response, its phenotype, and activation status, we isolated CD8+ T cells from tumors and secondary lymphoid organs and submitted them to T cell receptor (TCR) and total mRNA sequencing. We found an extremely diverse repertoire of more than 40 000 unique TCR sequences, but the ten most abundant TCRs accounted for >60% of CD8+ T-cell clones in the tumor. AH1-specific TCRs were consistently found among the most abundant sequences. AH1-specific T cells in the tumor had a tissue-resident memory phenotype. Treatment with L19-mIL12 led to overexpression of IL-12 receptor and of markers of cell activation and proliferation. These data suggest that the antitumor response driven by antibody-cytokine fusions proceeds through an oligoclonal expansion and activation of tumor-infiltrating CD8+ T cells.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Colon/patología , Neoplasias del Colon/terapia , Inmunoterapia/métodos , Virus de la Leucemia Murina/genética , Animales , Antígenos de Neoplasias/inmunología , Línea Celular Tumoral , Neoplasias del Colon/inmunología , Modelos Animales de Enfermedad , Femenino , Humanos , Memoria Inmunológica , Interleucina-12/uso terapéutico , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Péptidos/genética , Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/genética , Proteínas Oncogénicas de Retroviridae/genética , Proteínas Oncogénicas de Retroviridae/inmunología , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología
15.
Cancer Immunol Immunother ; 70(11): 3183-3197, 2021 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-33796916

RESUMEN

There is a growing interest in the use of patient-derived T cells for the treatment of various types of malignancies. The expansion of a polyclonal and polyspecific population of tumor-reactive T cells, with a subsequent infusion into the same donor patient, has been implemented, sometimes with positive results. It is not known, however, whether a set of T cells with a single antigen specificity may be sufficient for an effective therapy. To gain more insights in this matter, we used naturally occurring T cells recognizing a retroviral peptide (AH1), which is endogenous in many tumor cell lines of BALB/c origin and which serves as potent tumor rejection antigen. We were able to isolate and expand this rare population of T cells to numbers suitable for therapy experiments in mice (i.e., up to 30 × 106 cells/mouse). After the expansion process, T cells efficiently killed antigen-positive tumor cells in vitro and demonstrated tumor growth inhibition in two syngeneic murine models of cancer. However, AH1-specific T cells failed to induce complete regressions of established tumors. The incomplete activity was associated with a failure of injected T cells to survive in vivo, as only a very limited amount of T cells was found in tumor or secondary lymphoid organs 72 h after injection. These data suggest that future therapeutic strategies based on autologous T cells may require the potentiation of tumor-homing and survival properties of cancer-specific T cells.


Asunto(s)
Antígenos de Neoplasias/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/trasplante , Inmunoterapia Adoptiva/métodos , Neoplasias Experimentales/inmunología , Animales , Femenino , Ratones , Ratones Endogámicos BALB C , Proteínas de los Retroviridae/inmunología
16.
Chemistry ; 27(24): 7160-7167, 2021 Apr 26.
Artículo en Inglés | MEDLINE | ID: mdl-33586277

RESUMEN

The synthesis and characterization of a novel DNA-encoded library of macrocyclic peptide derivatives are described; the macrocycles are based on three sets of proteinogenic and non-proteinogenic amino acid building blocks and featuring the use of copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction for ring closure. The library (termed YO-DEL) which contains 1 254 838 compounds, was encoded with DNA in single-stranded format and was screened against target proteins of interest using affinity capture procedures and photocrosslinking. YO-DEL selections yielded specific binders against serum albumins, carbonic anhydrases and NKp46, a marker of activated Natural Killer cells.


Asunto(s)
Anhidrasas Carbónicas , Bibliotecas de Moléculas Pequeñas , ADN , Biblioteca de Genes , Péptidos
17.
Chemistry ; 27(35): 8985-8993, 2021 Jun 21.
Artículo en Inglés | MEDLINE | ID: mdl-33905156

RESUMEN

DNA-encoded chemical libraries are typically screened against purified protein targets. Recently, cell-based selections with encoded chemical libraries have been described, commonly revealing suboptimal performance due to insufficient recovery of binding molecules. We used carbonic anhydrase IX (CAIX)-expressing tumor cells as a model system to optimize selection procedures with code-specific quantitative polymerase chain reaction (qPCR) as selection readout. Salt concentration and performing PCR on cell suspension had the biggest impact on selection performance, leading to 15-fold enrichment factors for high-affinity monovalent CAIX binders (acetazolamide; KD =8.7 nM). Surprisingly, the homobivalent display of acetazolamide at the extremities of both complementary DNA strands led to a substantial improvement of both ligand recovery and enrichment factors (above 100-fold). The optimized procedures were used for selections with a DNA-encoded chemical library comprising 1 million members against tumor cell lines expressing CAIX, leading to a preferential recovery of known and new ligands against this validated tumor-associated target. This work may facilitate future affinity selections on cells against target proteins which might be difficult to express otherwise.


Asunto(s)
Anhidrasa Carbónica IX , ADN , Bibliotecas de Moléculas Pequeñas , Antígenos de Neoplasias/genética , Anhidrasa Carbónica IX/genética , Línea Celular Tumoral , Biblioteca de Genes , Humanos , Ligandos
18.
Bioorg Med Chem ; 41: 116206, 2021 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-34038862

RESUMEN

Progress in DNA-encoded chemical library synthesis and screening crucially relies on the availability of DNA-compatible reactions, which proceed with high yields and excellent purity for a large number of possible building blocks. In the past, experimental conditions have been presented for the execution of Suzuki and Sonogashira cross-coupling reactions on-DNA. In this article, our aim was to optimize Suzuki and Sonogashira reactions, comparing our results to previously published procedures. We have tested the performance of improved conditions using 606 building blocks (including boronic acids, pinacol boranes and terminal alkynes), achieving >70% conversion for 84% of the tested molecules. Moreover, we describe efficient experimental conditions for the on-DNA synthesis of amide bonds, starting from DNA derivatives carrying a carboxylic acid moiety and 300 primary, secondary and aromatic amines, as amide bonds are frequently found in DNA-encoded chemical libraries thanks to their excellent DNA compatibility.


Asunto(s)
Técnicas Químicas Combinatorias/métodos , ADN/química , Descubrimiento de Drogas/métodos , Bibliotecas de Moléculas Pequeñas , Amidas/química , Catálisis , Estructura Molecular
19.
Nanomedicine ; 36: 102424, 2021 08.
Artículo en Inglés | MEDLINE | ID: mdl-34174417

RESUMEN

In order to generate an antibody directed enzyme prodrug therapy, here we designed a chimeric protein by fusing the F8 antibody that recognizes the EDA of fibronectin (expressed on the tumor neovasculature) and an evolved variant of the ROS-generating enzyme D-amino acid oxidase (DAAO). The F8(scFv)-DAAO-Q144R recombinant protein is expressed by both CHO-S and E. coli cells. The F8(scFv)-DAAO-Q144R from E. coli cells is fully soluble, shows a high specific activity, is more thermostable in blood than the native DAAO, possesses a binding affinity for EDA well suited for efficient tumor accumulation, and localizes in tumor tissues. Notably, the F8(scFv)-DAAO-Q144R conjugate generates a stronger cytotoxicity to tumor cells than the native enzyme, especially when an inhibitor of heme oxygenase-1 (HO-1) is used, making it a promising candidate for a selective antitumor oxidative therapy controlled by the substrate addition, in the so called "activity on demand", thus sparing normal tissue from damage.


Asunto(s)
Anticuerpos Monoclonales Humanizados , Antineoplásicos , Citotoxinas , D-Aminoácido Oxidasa , Fibronectinas , Proteínas de Neoplasias , Neoplasias/tratamiento farmacológico , Proteínas Recombinantes de Fusión , Anticuerpos de Cadena Única , Animales , Anticuerpos Monoclonales Humanizados/química , Anticuerpos Monoclonales Humanizados/genética , Anticuerpos Monoclonales Humanizados/farmacología , Antineoplásicos/química , Antineoplásicos/farmacología , Células CHO , Células COS , Chlorocebus aethiops , Cricetulus , Citotoxinas/química , Citotoxinas/farmacología , D-Aminoácido Oxidasa/química , D-Aminoácido Oxidasa/genética , D-Aminoácido Oxidasa/farmacología , Fibronectinas/antagonistas & inhibidores , Fibronectinas/genética , Fibronectinas/metabolismo , Humanos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/farmacología
20.
Int J Mol Sci ; 22(18)2021 Sep 16.
Artículo en Inglés | MEDLINE | ID: mdl-34576184

RESUMEN

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.


Asunto(s)
Receptores del Factor de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales Humanizados/genética , Anticuerpos Monoclonales Humanizados/metabolismo , Cricetulus , Citocinas/genética , Citocinas/metabolismo , Femenino , Fibronectinas/genética , Fibronectinas/metabolismo , Técnica del Anticuerpo Fluorescente , Inmunoterapia , Ratones Endogámicos BALB C , Mutación , Estructura Secundaria de Proteína , Receptores del Factor de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/genética
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