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1.
Cell ; 185(10): 1745-1763.e22, 2022 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-35483375

RESUMO

Regulatable CAR platforms could circumvent toxicities associated with CAR-T therapy, but existing systems have shortcomings including leakiness and attenuated activity. Here, we present SNIP CARs, a protease-based platform for regulating CAR activity using an FDA-approved small molecule. Design iterations yielded CAR-T cells that manifest full functional capacity with drug and no leaky activity in the absence of drug. In numerous models, SNIP CAR-T cells were more potent than constitutive CAR-T cells and showed diminished T cell exhaustion and greater stemness. In a ROR1-based CAR lethality model, drug cessation following toxicity onset reversed toxicity, thereby credentialing the platform as a safety switch. In the same model, reduced drug dosing opened a therapeutic window that resulted in tumor eradication in the absence of toxicity. SNIP CARs enable remote tuning of CAR activity, which provides solutions to safety and efficacy barriers that are currently limiting progress in using CAR-T cells to treat solid tumors.


Assuntos
Neoplasias , Receptores de Antígenos Quiméricos , Humanos , Imunoterapia Adotiva/métodos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Peptídeo Hidrolases , Receptores de Antígenos de Linfócitos T , Linfócitos T/patologia
2.
Nature ; 630(8016): 457-465, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38750365

RESUMO

Adoptively transferred T cells and agents designed to block the CD47-SIRPα axis are promising cancer therapeutics that activate distinct arms of the immune system1,2. Here we administered anti-CD47 antibodies in combination with adoptively transferred T cells with the goal of enhancing antitumour efficacy but observed abrogated therapeutic benefit due to rapid macrophage-mediated clearance of T cells expressing chimeric antigen receptors (CARs) or engineered T cell receptors. Anti-CD47-antibody-mediated CAR T cell clearance was potent and rapid enough to serve as an effective safety switch. To overcome this challenge, we engineered the CD47 variant CD47(Q31P) (47E), which engages SIRPα and provides a 'don't eat me' signal that is not blocked by anti-CD47 antibodies. TCR or CAR T cells expressing 47E are resistant to clearance by macrophages after treatment with anti-CD47 antibodies, and mediate substantial, sustained macrophage recruitment to the tumour microenvironment. Although many of the recruited macrophages manifested an M2-like profile3, the combined therapy synergistically enhanced antitumour efficacy. Our study identifies macrophages as major regulators of T cell persistence and illustrates the fundamental challenge of combining T-cell-directed therapeutics with those designed to activate macrophages. It delivers a therapeutic approach that is capable of simultaneously harnessing the antitumour effects of T cells and macrophages, offering enhanced potency against solid tumours.


Assuntos
Antígeno CD47 , Imunoterapia Adotiva , Neoplasias , Linfócitos T , Animais , Feminino , Humanos , Masculino , Camundongos , Antígenos de Diferenciação/imunologia , Antígenos de Diferenciação/metabolismo , Antígeno CD47/genética , Antígeno CD47/imunologia , Antígeno CD47/metabolismo , Linhagem Celular Tumoral , Imunoterapia Adotiva/métodos , Macrófagos/citologia , Macrófagos/imunologia , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/transplante , Microambiente Tumoral/imunologia , Anticorpos/imunologia , Anticorpos/uso terapêutico , Ativação de Macrófagos
3.
Proc Natl Acad Sci U S A ; 121(12): e2310866121, 2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38483996

RESUMO

Lymphocyte activation gene-3 (LAG-3) is an inhibitory receptor expressed on activated T cells and an emerging immunotherapy target. Domain 1 (D1) of LAG-3, which has been purported to directly interact with major histocompatibility complex class II (MHCII) and fibrinogen-like protein 1 (FGL1), has been the major focus for the development of therapeutic antibodies that inhibit LAG-3 receptor-ligand interactions and restore T cell function. Here, we present a high-resolution structure of glycosylated mouse LAG-3 ectodomain, identifying that cis-homodimerization, mediated through a network of hydrophobic residues within domain 2 (D2), is critically required for LAG-3 function. Additionally, we found a previously unidentified key protein-glycan interaction in the dimer interface that affects the spatial orientation of the neighboring D1 domain. Mutation of LAG-3 D2 residues reduced dimer formation, dramatically abolished LAG-3 binding to both MHCII and FGL1 ligands, and consequentially inhibited the role of LAG-3 in suppressing T cell responses. Intriguingly, we showed that antibodies directed against D1, D2, and D3 domains are all capable of blocking LAG-3 dimer formation and MHCII and FGL-1 ligand binding, suggesting a potential allosteric model of LAG-3 function tightly regulated by dimerization. Furthermore, our work reveals unique epitopes, in addition to D1, that can be targeted for immunotherapy of cancer and other human diseases.


Assuntos
Antígenos de Histocompatibilidade Classe II , Linfócitos T , Animais , Humanos , Camundongos , Dimerização , Fibrinogênio/metabolismo , Ligantes , Mutação
4.
J Immunol ; 211(2): 295-305, 2023 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-37256255

RESUMO

Spontaneous tumors that arise in genetically engineered mice recapitulate the natural tumor microenvironment and tumor-immune coevolution observed in human cancers, providing a more physiologically relevant preclinical model relative to implanted tumors. Similar to many cancer patients, oncogene-driven spontaneous tumors are often resistant to immunotherapy, and thus novel agents that can effectively promote antitumor immunity against these aggressive cancers show considerable promise for clinical translation, and their mechanistic assessment can broaden our understanding of tumor immunology. In this study, we performed extensive immune profiling experiments to investigate how tumor-targeted TLR9 stimulation remodels the microenvironment of spontaneously arising tumors during an effective antitumor immune response. To model the clinical scenario of multiple tumor sites, we used MMTV-PyMT transgenic mice, which spontaneously develop heterogeneous breast tumors throughout their 10 mammary glands. We found that i.v. administration of a tumor-targeting TLR9 agonist, referred to as PIP-CpG, induced a systemic T cell-mediated immune response that not only promoted regression of existing mammary tumors, but also elicited immune memory capable of delaying growth of independent newly arising tumors. Within the tumor microenvironment, PIP-CpG therapy initiated an inflammatory cascade that dramatically amplified chemokine and cytokine production, prompted robust infiltration and expansion of innate and adaptive immune cells, and led to diverse and unexpected changes in immune phenotypes. This study demonstrates that effective systemic treatment of an autochthonous multisite tumor model can be achieved using a tumor-targeted immunostimulant and provides immunological insights that will inform future therapeutic strategies.


Assuntos
Neoplasias da Mama , Neoplasias Mamárias Animais , Camundongos , Animais , Humanos , Feminino , Receptor Toll-Like 9 , Camundongos Transgênicos , Adjuvantes Imunológicos/farmacologia , Neoplasias Mamárias Animais/terapia , Neoplasias da Mama/terapia , Microambiente Tumoral , Linhagem Celular Tumoral
5.
J Biol Chem ; 299(6): 104755, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37116708

RESUMO

The colony-stimulating factor 3 receptor (CSF3R) controls the growth of neutrophils, the most abundant type of white blood cell. In healthy neutrophils, signaling is dependent on CSF3R binding to its ligand, CSF3. A single amino acid mutation in CSF3R, T618I, instead allows for constitutive, ligand-independent cell growth and leads to a rare type of cancer called chronic neutrophilic leukemia. However, the disease mechanism is not well understood. Here, we investigated why this threonine to isoleucine substitution is the predominant mutation in chronic neutrophilic leukemia and how it leads to uncontrolled neutrophil growth. Using protein domain mapping, we demonstrated that the single CSF3R domain containing residue 618 is sufficient for ligand-independent activity. We then applied an unbiased mutational screening strategy focused on this domain and found that activating mutations are enriched at sites normally occupied by asparagine, threonine, and serine residues-the three amino acids which are commonly glycosylated. We confirmed glycosylation at multiple CSF3R residues by mass spectrometry, including the presence of GalNAc and Gal-GalNAc glycans at WT threonine 618. Using the same approach applied to other cell surface receptors, we identified an activating mutation, S489F, in the interleukin-31 receptor alpha chain. Combined, these results suggest a role for glycosylated hotspot residues in regulating receptor signaling, mutation of which can lead to ligand-independent, uncontrolled activity and human disease.


Assuntos
Leucemia Neutrofílica Crônica , Humanos , Leucemia Neutrofílica Crônica/diagnóstico , Leucemia Neutrofílica Crônica/genética , Leucemia Neutrofílica Crônica/metabolismo , Glicosilação , Ligantes , Mutação , Receptores de Fator Estimulador de Colônias/genética , Receptores de Fator Estimulador de Colônias/metabolismo , Treonina/metabolismo , Fatores Estimuladores de Colônias/genética , Fatores Estimuladores de Colônias/metabolismo
6.
Biotechnol Bioeng ; 121(10): 3169-3180, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-38965775

RESUMO

Urokinase-type plasminogen activator receptor (uPAR) is overexpressed on tumor cells in multiple types of cancer and contributes to disease progression and metastasis. In this work, we engineered a novel bi-paratopic uPAR targeting agent by fusing the binding domains of two native uPAR ligands: uPA and vitronectin, with a flexible peptide linker. The linker length was optimized to facilitate simultaneous engagement of both domains to their adjacent epitopes on uPAR, resulting in a high affinity and avid binding interaction. Furthermore, the individual domains were affinity-matured using yeast surface display and directed evolution, resulting in a bi-paratopic protein with affinity in the picomolar to femtomolar range. This engineered uPAR targeting agent demonstrated significantly enhanced tumor localization in mouse tumor models compared to the native uPAR ligand and warrants further investigation as a diagnostic and therapeutic agent for cancer.


Assuntos
Receptores de Ativador de Plasminogênio Tipo Uroquinase , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Animais , Camundongos , Humanos , Engenharia de Proteínas/métodos , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/genética , Linhagem Celular Tumoral , Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Vitronectina/metabolismo , Vitronectina/química , Vitronectina/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/química
7.
Nat Methods ; 17(8): 852-860, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32661427

RESUMO

Sensitive detection of two biological events in vivo has long been a goal in bioluminescence imaging. Antares, a fusion of the luciferase NanoLuc to the orange fluorescent protein CyOFP, has emerged as a bright bioluminescent reporter with orthogonal substrate specificity to firefly luciferase (FLuc) and its derivatives such as AkaLuc. However, the brightness of Antares in mice is limited by the poor solubility and bioavailability of the NanoLuc substrate furimazine. Here, we report a new substrate, hydrofurimazine, whose enhanced aqueous solubility allows delivery of higher doses to mice. In the liver, Antares with hydrofurimazine exhibited similar brightness to AkaLuc with its substrate AkaLumine. Further chemical exploration generated a second substrate, fluorofurimazine, with even higher brightness in vivo. We used Antares with fluorofurimazine to track tumor size and AkaLuc with AkaLumine to visualize CAR-T cells within the same mice, demonstrating the ability to perform two-population imaging with these two luciferase systems.


Assuntos
Furanos/química , Luciferases/química , Medições Luminescentes/métodos , Proteínas Luminescentes/química , Animais , Ensaios Enzimáticos/métodos , Especificidade por Substrato
8.
Nat Chem Biol ; 17(9): 937-946, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33767387

RESUMO

Selective protein degradation platforms have afforded new development opportunities for therapeutics and tools for biological inquiry. The first lysosome-targeting chimeras (LYTACs) targeted extracellular and membrane proteins for degradation by bridging a target protein to the cation-independent mannose-6-phosphate receptor (CI-M6PR). Here, we developed LYTACs that engage the asialoglycoprotein receptor (ASGPR), a liver-specific lysosome-targeting receptor, to degrade extracellular proteins in a cell-type-specific manner. We conjugated binders to a triantenerrary N-acetylgalactosamine (tri-GalNAc) motif that engages ASGPR to drive the downregulation of proteins. Degradation of epidermal growth factor receptor (EGFR) by GalNAc-LYTAC attenuated EGFR signaling compared to inhibition with an antibody. Furthermore, we demonstrated that a LYTAC consisting of a 3.4-kDa peptide binder linked to a tri-GalNAc ligand degrades integrins and reduces cancer cell proliferation. Degradation with a single tri-GalNAc ligand prompted site-specific conjugation on antibody scaffolds, which improved the pharmacokinetic profile of GalNAc-LYTACs in vivo. GalNAc-LYTACs thus represent an avenue for cell-type-restricted protein degradation.


Assuntos
Receptor de Asialoglicoproteína/metabolismo , Lisossomos/metabolismo , Acetilgalactosamina/metabolismo , Humanos , Células Tumorais Cultivadas
9.
Proc Natl Acad Sci U S A ; 117(25): 14110-14118, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32522868

RESUMO

Interleukin-6 (IL-6) family cytokines signal through multimeric receptor complexes, providing unique opportunities to create novel ligand-based therapeutics. The cardiotrophin-like cytokine factor 1 (CLCF1) ligand has been shown to play a role in cancer, osteoporosis, and atherosclerosis. Once bound to ciliary neurotrophic factor receptor (CNTFR), CLCF1 mediates interactions to coreceptors glycoprotein 130 (gp130) and leukemia inhibitory factor receptor (LIFR). By increasing CNTFR-mediated binding to these coreceptors we generated a receptor superagonist which surpassed the potency of natural CNTFR ligands in neuronal signaling. Through additional mutations, we generated a receptor antagonist with increased binding to CNTFR but lack of binding to the coreceptors that inhibited tumor progression in murine xenograft models of nonsmall cell lung cancer. These studies further validate the CLCF1-CNTFR signaling axis as a therapeutic target and highlight an approach of engineering cytokine activity through a small number of mutations.


Assuntos
Subunidade alfa do Receptor do Fator Neutrófico Ciliar/agonistas , Citocinas/metabolismo , Engenharia de Proteínas/métodos , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Células Cultivadas , Subunidade alfa do Receptor do Fator Neutrófico Ciliar/antagonistas & inibidores , Subunidade alfa do Receptor do Fator Neutrófico Ciliar/metabolismo , Receptor gp130 de Citocina/metabolismo , Citocinas/química , Citocinas/genética , Humanos , Subunidade alfa de Receptor de Fator Inibidor de Leucemia/metabolismo , Ligantes , Neurônios/metabolismo , Ligação Proteica , Ratos , Transdução de Sinais
10.
Cytokine ; 127: 154974, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31978642

RESUMO

Although ischemic heart disease is the leading cause of death worldwide, mainstay treatments ultimately fail because they do not adequately address disease pathophysiology. Restoring the microvascular perfusion deficit remains a significant unmet need and may be addressed via delivery of pro-angiogenic cytokines. The therapeutic effect of cytokines can be enhanced by encapsulation within hydrogels, but current hydrogels do not offer sufficient clinical translatability due to unfavorable viscoelastic mechanical behavior which directly impacts the ability for minimally-invasive catheter delivery. In this report, we examine the therapeutic implications of dual-stage cytokine release from a novel, highly shear-thinning biocompatible catheter-deliverable hydrogel. We chose to encapsulate two protein-engineered cytokines, namely dimeric fragment of hepatocyte growth factor (HGFdf) and engineered stromal cell-derived factor 1α (ESA), which target distinct disease pathways. The controlled release of HGFdf and ESA from separate phases of the hyaluronic acid-based hydrogel allows extended and pronounced beneficial effects due to the precise timing of release. We evaluated the therapeutic efficacy of this treatment strategy in a small animal model of myocardial ischemia and observed a significant benefit in biological and functional parameters. Given the encouraging results from the small animal experiment, we translated this treatment to a large animal preclinical model and observed a reduction in scar size, indicating this strategy could serve as a potential adjunct therapy for the millions of people suffering from ischemic heart disease.


Assuntos
Hidrogéis/administração & dosagem , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Função Ventricular Esquerda/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacos , Animais , Catéteres , Células Cultivadas , Modelos Animais de Doenças , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Ácido Hialurônico/administração & dosagem , Isquemia Miocárdica/tratamento farmacológico , Isquemia Miocárdica/metabolismo , Miocárdio/patologia , Ratos
11.
J Biol Chem ; 293(14): 4969-4980, 2018 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-29386351

RESUMO

Dysregulated matriptase activity has been established as a key contributor to cancer progression through its activation of growth factors, including the hepatocyte growth factor (HGF). Despite its critical role and prevalence in many human cancers, limitations to developing an effective matriptase inhibitor include weak binding affinity, poor selectivity, and short circulating half-life. We applied rational and combinatorial approaches to engineer a potent inhibitor based on the hepatocyte growth factor activator inhibitor type-1 (HAI-1), a natural matriptase inhibitor. The first Kunitz domain (KD1) of HAI-1 has been well established as a minimal matriptase-binding and inhibition domain, whereas the second Kunitz domain (KD2) is inactive and involved in negative regulation. Here, we replaced the inactive KD2 domain of HAI-1 with an engineered chimeric variant of KD2/KD1 domains and fused the resulting construct to an antibody Fc domain to increase valency and circulating serum half-life. The final protein variant contains four stoichiometric binding sites that we showed were needed to effectively inhibit matriptase with a Ki of 70 ± 5 pm, an increase of 120-fold compared with the natural HAI-1 inhibitor, to our knowledge making it one of the most potent matriptase inhibitors identified to date. Furthermore, the engineered inhibitor demonstrates a protease selectivity profile similar to that of wildtype KD1 but distinct from that of HAI-1. It also inhibits activation of the natural pro-HGF substrate and matriptase expressed on cancer cells with at least an order of magnitude greater efficacy than KD1.


Assuntos
Engenharia de Proteínas/métodos , Proteínas Secretadas Inibidoras de Proteinases/química , Proteínas Secretadas Inibidoras de Proteinases/genética , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacologia , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Cães , Humanos , Células Madin Darby de Rim Canino , Domínios Proteicos , Proteínas Secretadas Inibidoras de Proteinases/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia
12.
Nucleic Acids Res ; 45(7): 3615-3626, 2017 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-28334756

RESUMO

Our understanding of translation underpins our capacity to engineer living systems. The canonical start codon (AUG) and a few near-cognates (GUG, UUG) are considered as the 'start codons' for translation initiation in Escherichia coli. Translation is typically not thought to initiate from the 61 remaining codons. Here, we quantified translation initiation of green fluorescent protein and nanoluciferase in E. coli from all 64 triplet codons and across a range of DNA copy number. We detected initiation of protein synthesis above measurement background for 47 codons. Translation from non-canonical start codons ranged from 0.007 to 3% relative to translation from AUG. Translation from 17 non-AUG codons exceeded the highest reported rates of non-cognate codon recognition. Translation initiation from non-canonical start codons may contribute to the synthesis of peptides in both natural and synthetic biological systems.


Assuntos
Códon de Iniciação , Escherichia coli/genética , Iniciação Traducional da Cadeia Peptídica , Códon , Proteínas de Fluorescência Verde/genética , Luciferases/genética , Plasmídeos/genética
13.
Nat Chem Biol ; 12(2): 76-81, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26641932

RESUMO

We describe a multipurpose technology platform, termed µSCALE (microcapillary single-cell analysis and laser extraction), that enables massively parallel, quantitative biochemical and biophysical measurements on millions of protein variants expressed from yeast or bacteria. µSCALE spatially segregates single cells within a microcapillary array, enabling repeated imaging, cell growth and protein expression. We performed high-throughput analysis of cells and their protein products using a range of fluorescent assays, including binding-affinity measurements and dynamic enzymatic assays. A precise laser-based extraction method allows rapid recovery of live clones and their genetic material from microcapillaries for further study. With µSCALE, we discovered a new antibody against a clinical cancer target, evolved a fluorescent protein biosensor and engineered an enzyme to reduce its sensitivity to its inhibitor. These protein analysis and engineering applications each have unique assay requirements and different host organisms, highlighting the flexibility and technical capabilities of the µSCALE platform.


Assuntos
Proteínas de Bactérias/análise , Técnicas de Química Analítica/instrumentação , Proteínas Fúngicas/análise , Análise Serial de Proteínas/instrumentação , Engenharia de Proteínas/instrumentação , Análise de Célula Única/instrumentação , Técnicas Biossensoriais/instrumentação , Citometria de Fluxo , Corantes Fluorescentes/química , Biblioteca Gênica , Ligação Proteica
14.
Biochemistry ; 56(43): 5720-5725, 2017 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-28952732

RESUMO

Homochirality is a general feature of biological macromolecules, and Nature includes few examples of heterochiral proteins. Herein, we report on the design, chemical synthesis, and structural characterization of heterochiral proteins possessing loops of amino acids of chirality opposite to that of the rest of a protein scaffold. Using the protein Ecballium elaterium trypsin inhibitor II, we discover that selective ß-alanine substitution favors the efficient folding of our heterochiral constructs. Solution nuclear magnetic resonance spectroscopy of one such heterochiral protein reveals a homogeneous global fold. Additionally, steered molecular dynamics simulation indicate ß-alanine reduces the free energy required to fold the protein. We also find these heterochiral proteins to be more resistant to proteolysis than homochiral l-proteins. This work informs the design of heterochiral protein architectures containing stretches of both d- and l-amino acids.


Assuntos
Cucurbitaceae/química , Simulação de Dinâmica Molecular , Proteínas de Plantas/química , Dobramento de Proteína , Cucurbitaceae/genética , Proteínas de Plantas/genética , Domínios Proteicos
15.
Biotechnol Bioeng ; 114(10): 2379-2389, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28574594

RESUMO

In the last decade, numerous growth factors and biomaterials have been explored for the treatment of myocardial infarction (MI). While pre-clinical studies have demonstrated promising results, clinical trials have been disappointing and inconsistent, likely due to poor translatability. In the present study, we investigate a potential myocardial regenerative therapy consisting of a protein-engineered dimeric fragment of hepatocyte growth factor (HGFdf) encapsulated in a shear-thinning, self-healing, bioengineered hydrogel (SHIELD). We hypothesized that SHIELD would facilitate targeted, sustained intramyocardial delivery of HGFdf thereby attenuating myocardial injury and post-infarction remodeling. Adult male Wistar rats (n = 45) underwent sham surgery or induction of MI followed by injection of phosphate buffered saline (PBS), 10 µg HGFdf alone, SHIELD alone, or SHIELD encapsulating 10 µg HGFdf. Ventricular function, infarct size, and angiogenic response were assessed 4 weeks post-infarction. Treatment with SHIELD + HGFdf significantly reduced infarct size and increased both ejection fraction and borderzone arteriole density compared to the controls. Thus, sustained delivery of HGFdf via SHIELD limits post-infarction adverse ventricular remodeling by increasing angiogenesis and reducing fibrosis. Encapsulation of HGFdf in SHIELD improves clinical translatability by enabling minimally-invasive delivery and subsequent retention and sustained administration of this novel, potent angiogenic protein analog. Biotechnol. Bioeng. 2017;114: 2379-2389. © 2017 Wiley Periodicals, Inc.


Assuntos
Preparações de Ação Retardada/administração & dosagem , Fator de Crescimento de Hepatócito/administração & dosagem , Hidrogéis/química , Infarto do Miocárdio/tratamento farmacológico , Engenharia de Proteínas/métodos , Proteínas Recombinantes/administração & dosagem , Disfunção Ventricular Esquerda/prevenção & controle , Proteínas Angiogênicas/administração & dosagem , Proteínas Angiogênicas/química , Proteínas Angiogênicas/genética , Animais , Preparações de Ação Retardada/química , Difusão , Fator de Crescimento de Hepatócito/análogos & derivados , Fator de Crescimento de Hepatócito/genética , Injeções , Masculino , Infarto do Miocárdio/diagnóstico por imagem , Infarto do Miocárdio/patologia , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Resistência ao Cisalhamento , Resultado do Tratamento , Disfunção Ventricular Esquerda/diagnóstico por imagem , Disfunção Ventricular Esquerda/patologia , Viscosidade
16.
J Am Chem Soc ; 138(24): 7496-9, 2016 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-27280683

RESUMO

Chemoenzymatic modification of proteins is an attractive option to create highly specific conjugates for therapeutics, diagnostics, or materials under gentle biological conditions. However, these methods often suffer from expensive specialized substrates, bulky fusion tags, low yields, and extra purification steps to achieve the desired conjugate. Staphylococcus aureus sortase A and its engineered variants are used to attach oligoglycine derivatives to the C-terminus of proteins expressed with a minimal LPXTG tag. This strategy has been used extensively for bioconjugation in vitro and for protein-protein conjugation in living cells. Here we show that an enzyme variant recently engineered for higher activity on oligoglycine has promiscuous activity that allows proteins to be tagged using a diverse array of small, commercially available amines, including several bioorthogonal functional groups. This technique can also be carried out in living Escherichia coli, enabling simple, inexpensive production of chemically functionalized proteins with no additional purification steps.


Assuntos
Aminas/química , Aminoaciltransferases/química , Proteínas de Bactérias/química , Cisteína Endopeptidases/química , Glicina/química , Engenharia de Proteínas/métodos , Staphylococcus aureus/enzimologia , Aminoaciltransferases/genética , Proteínas de Bactérias/genética , Cisteína Endopeptidases/genética , Escherichia coli/genética , Estrutura Molecular
17.
Nat Chem Biol ; 10(11): 977-83, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25242553

RESUMO

Aberrant signaling through the Axl receptor tyrosine kinase has been associated with a myriad of human diseases, most notably metastatic cancer, identifying Axl and its ligand Gas6 as important therapeutic targets. Using rational and combinatorial approaches, we engineered an Axl 'decoy receptor' that binds Gas6 with high affinity and inhibits its function, offering an alternative approach from drug discovery efforts that directly target Axl. Four mutations within this high-affinity Axl variant caused structural alterations in side chains across the Gas6-Axl binding interface, stabilizing a conformational change on Gas6. When reformatted as an Fc fusion, the engineered decoy receptor bound Gas6 with femtomolar affinity, an 80-fold improvement compared to binding of the wild-type Axl receptor, allowing effective sequestration of Gas6 and specific abrogation of Axl signaling. Moreover, increased Gas6 binding affinity was critical and correlative with the ability of decoy receptors to potently inhibit metastasis and disease progression in vivo.


Assuntos
Engenharia Genética , Fragmentos Fc das Imunoglobulinas/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/farmacologia , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Sítios de Ligação , Progressão da Doença , Relação Dose-Resposta a Droga , Humanos , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/isolamento & purificação , Fragmentos Fc das Imunoglobulinas/uso terapêutico , Peptídeos e Proteínas de Sinalização Intercelular/química , Camundongos , Modelos Moleculares , Mutação/genética , Metástase Neoplásica/tratamento farmacológico , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Relação Estrutura-Atividade , Receptor Tirosina Quinase Axl
18.
Proc Natl Acad Sci U S A ; 110(36): 14598-603, 2013 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-23950221

RESUMO

Central nervous system tumors carry grave clinical prognoses due to limited effectiveness of surgical resection, radiation, and chemotherapy. Thus, improved strategies for brain tumor visualization and targeted treatment are critically needed. We demonstrate that mouse cerebellar medulloblastoma (MB) can be targeted and illuminated with a fluorescent, engineered cystine knot (knottin) peptide that binds with high affinity to αvß3, αvß5, and α5ß1 integrin receptors. This integrin-binding knottin peptide, denoted EETI 2.5F, was evaluated as a molecular imaging probe in both orthotopic and genetic models of MB. Following tail vein injection, fluorescence arising from dye-conjugated EETI 2.5F was localized to the tumor compared with the normal surrounding brain tissue, as measured by optical imaging. The imaging signal intensity correlated with tumor volume. Due to its unique ability to bind to α5ß1 integrin, EETI 2.5F showed superior in vivo and ex vivo brain tumor imaging contrast compared with other engineered integrin-binding knottin peptides and with c(RGDfK), a well-studied integrin-binding peptidomimetic. Next, EETI 2.5F was fused to an antibody fragment crystallizable (Fc) domain (EETI 2.5F-Fc) to determine if a larger integrin-binding protein could also target intracranial brain tumors. EETI 2.5F-Fc, conjugated to a fluorescent dye, illuminated MB following i.v. injection and was able to distribute throughout the tumor parenchyma. In contrast, brain tumor imaging signals were not detected in mice injected with EETI 2.5F proteins containing a scrambled integrin-binding sequence, demonstrating the importance of target specificity. These results highlight the potential of using EETI 2.5F and EETI 2.5-Fc as targeted molecular probes for brain tumor imaging.


Assuntos
Neoplasias Cerebelares/diagnóstico , Miniproteínas Nó de Cistina/metabolismo , Diagnóstico por Imagem/métodos , Meduloblastoma/diagnóstico , Animais , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/metabolismo , Miniproteínas Nó de Cistina/química , Miniproteínas Nó de Cistina/genética , Feminino , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Integrina alfa5beta1/metabolismo , Masculino , Meduloblastoma/genética , Meduloblastoma/metabolismo , Camundongos , Camundongos Knockout , Camundongos Nus , Camundongos Transgênicos , Microscopia de Fluorescência , Imagem Molecular/métodos , Receptores Patched , Ligação Proteica , Engenharia de Proteínas , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Sensibilidade e Especificidade
19.
Angew Chem Int Ed Engl ; 55(34): 9894-7, 2016 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-27304709

RESUMO

Antibody-drug conjugates (ADCs) offer increased efficacy and reduced toxicity compared to systemic chemotherapy. Less attention has been paid to peptide-drug delivery, which has the potential for increased tumor penetration and facile synthesis. We report a knottin peptide-drug conjugate (KDC) and demonstrate that it can selectively deliver gemcitabine to malignant cells expressing tumor-associated integrins. This KDC binds to tumor cells with low-nanomolar affinity, is internalized by an integrin-mediated process, releases its payload intracellularly, and is a highly potent inhibitor of brain, breast, ovarian, and pancreatic cancer cell lines. Notably, these features enable this KDC to bypass a gemcitabine-resistance mechanism found in pancreatic cancer cells. This work expands the therapeutic relevance of knottin peptides to include targeted drug delivery, and further motivates efforts to expand the drug-conjugate toolkit to include non-antibody protein scaffolds.


Assuntos
Antineoplásicos/farmacologia , Miniproteínas Nó de Cistina/metabolismo , Desoxicitidina/análogos & derivados , Integrinas/antagonistas & inibidores , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Miniproteínas Nó de Cistina/química , Desoxicitidina/química , Desoxicitidina/farmacologia , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Conformação Molecular , Relação Estrutura-Atividade , Gencitabina
20.
J Am Chem Soc ; 137(1): 6-9, 2015 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-25486381

RESUMO

Molecules that target and inhibit αvß3, αvß5, and α5ß1 integrins have generated great interest because of the role of these receptors in mediating angiogenesis and metastasis. Attempts to increase the binding affinity and hence the efficacy of integrin inhibitors by dimerization have been marginally effective. In the present work, we achieved this goal by using oxime-based chemical conjugation to synthesize dimers of integrin-binding cystine knot (knottin) miniproteins with low-picomolar binding affinity to tumor cells. A non-natural amino acid containing an aminooxy side chain was introduced at different locations within a knottin monomer and reacted with dialdehyde-containing cross-linkers of different lengths to create knottin dimers with varying molecular topologies. Dimers cross-linked through an aminooxy functional group located near the middle of the protein exhibited higher apparent binding affinity to integrin-expressing tumor cells compared with dimers cross-linked through an aminooxy group near the C-terminus. In contrast, the cross-linker length had no effect on the integrin binding affinity. A chemical-based dimerization strategy was critical, as knottin dimers created through genetic fusion to a bivalent antibody domain exhibited only modest improvement (less than 5-fold) in tumor cell binding relative to the knottin monomer. The best oxime-conjugated knottin dimer achieved an unprecedented 150-fold increase in apparent binding affinity over the knottin monomer. Also, this dimer bound 3650-fold stronger and inhibited tumor cell migration and proliferation compared with cilengitide, an integrin-targeting peptidomimetic that performed poorly in recent clinical trials, suggesting promise for further therapeutic development.


Assuntos
Movimento Celular/efeitos dos fármacos , Reagentes de Ligações Cruzadas/química , Miniproteínas Nó de Cistina/química , Miniproteínas Nó de Cistina/farmacologia , Integrinas/antagonistas & inibidores , Integrinas/química , Neoplasias/patologia , Multimerização Proteica , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Miniproteínas Nó de Cistina/síntese química , Relação Dose-Resposta a Droga , Humanos , Integrinas/metabolismo , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade
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