RESUMEN
Atherosclerosis is a chronic inflammatory disease associated with the accumulation of low-density lipoprotein (LDL) in arterial walls. Higher levels of the anti-inflammatory cytokine IL-10 in serum are correlated with reduced plaque burden. However, cytokine therapies have not translated well to the clinic, partially due to their rapid clearance and pleiotropic nature. Here, we engineered IL-10 to overcome these challenges by hitchhiking on LDL to atherosclerotic plaques. Specifically, we constructed fusion proteins in which one domain is IL-10 and the other is an antibody fragment (Fab) that binds to protein epitopes of LDL. In murine models of atherosclerosis, we show that systemically administered Fab-IL-10 constructs bind circulating LDL and traffic to atherosclerotic plaques. One such construct, 2D03-IL-10, significantly reduces aortic immune cell infiltration to levels comparable to healthy mice, whereas non-targeted IL-10 has no therapeutic effect. Mechanistically, we demonstrate that 2D03-IL-10 preferentially associates with foamy macrophages and reduces pro-inflammatory activation markers. This platform technology can be applied to a variety of therapeutics and shows promise as a potential targeted anti-inflammatory therapy in atherosclerosis.
RESUMEN
Cancer immunotherapy is moving toward combination regimens with agents of complementary mechanisms of action to achieve more frequent and robust efficacy. However, compared with single-agent therapies, combination immunotherapies are associated with increased overall toxicity because the very same mechanisms also work in concert to enhance systemic inflammation and promote off-tumor toxicity. Therefore, rational design of combination regimens that achieve improved antitumor control without exacerbated toxicity is a main objective in combination immunotherapy. Here, we show that the combination of engineered, tumor matrix-binding interleukin-7 (IL-7) and IL-12 achieves remarkable anticancer effects by activating complementary pathways without inducing any additive immunotoxicity. Mechanistically, engineered IL-12 provided effector properties to T cells, while IL-7 prevented their exhaustion and boosted memory formation as assessed by tumor rechallenge experiments. The dual combination also rendered checkpoint inhibitor (CPI)-resistant genetically engineered melanoma model responsive to CPI. Thus, our approach provides a framework of evaluation of rationally designed combinations in immuno-oncology and yields a promising therapy.
Asunto(s)
Interleucina-12 , Melanoma , Humanos , Interleucina-12/genética , Interleucina-7/farmacología , Agotamiento de Células T , Inmunoterapia , Melanoma/patologíaRESUMEN
The pathogenesis of cutaneous T-cell lymphoma (CTCL) remains unclear. Using single-cell RNA or T-cell receptor (TCR) sequencing of 32 619 CD3+CD4+ and CD26+/CD7+ and 29 932 CD3+CD4+ and CD26-/CD7- lymphocytes from the peripheral blood of 7 patients with CTCL, coupled to single-cell ATAC-sequencing of 26,411 CD3+CD4+ and CD26+/CD7+ and 33 841 CD3+CD4+ and CD26-/CD7- lymphocytes, we show that tumor cells in Sézary syndrome and mycosis fungoides (MF) exhibit different phenotypes and trajectories of differentiation. When compared to MF, Sézary cells exhibit narrower repertoires of TCRs and exhibit clonal enrichment. Surprisingly, we identified ≥200 mutations in hematopoietic stem cells from multiple patients with Sézary syndrome. Mutations in key oncogenes were also present in peripheral Sézary cells, which also showed the hallmarks of recent thymic egression. Together our data suggest that CTCL arises from mutated lymphocyte progenitors that acquire TCRs in the thymus, which complete their malignant transformation in the periphery.
Asunto(s)
Linfoma Cutáneo de Células T , Micosis Fungoide , Síndrome de Sézary , Neoplasias Cutáneas , Humanos , Síndrome de Sézary/genética , Síndrome de Sézary/patología , Dipeptidil Peptidasa 4 , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Micosis Fungoide/genética , Micosis Fungoide/patología , Linfoma Cutáneo de Células T/genética , Receptores de Antígenos de Linfocitos TRESUMEN
PURPOSE: Immune checkpoint blockade holds promise for treating bacillus Calmette-Guerin (BCG)-unresponsive non-muscle-invasive bladder cancer (NMIBC). In this phase II study, we investigated the safety and efficacy of durvalumab, a human IgG1 monoclonal antibody, against BCG-unresponsive carcinoma in situ (CIS). PATIENTS AND METHODS: Patients with BCG-unresponsive CIS-containing NMIBC received durvalumab IV at 1,500 mg every 4 weeks for up to 12 months. The primary endpoint was complete response (CR) rate at month 6, defined by negative cystoscopy, urine cytology, and absence of high-grade recurrence on bladder mapping biopsy. The null hypothesis specified a CR rate of 18% and alternative hypothesis of 40%. According to the Simon two-stage design, if ≤3/13 patients achieved CR during stage 1, the trial is stopped due to futility. RESULTS: Between March 8, 2017, and January 24, 2020, 17 patients were accrued whereas 4 withdrew from study treatment after bladder biopsy at month 3 was positive for CIS. Two of 17 (12%) achieved a CR at month 6, with duration of response of 10 and 18 months, respectively. A single grade 3 lipase elevation was attributed to durvalumab, and immune-related adverse events were observed in 7/17 (41%) patients. Only 1/17 patients had high programmed death-ligand 1 expression pretreatment. On RNA sequencing, complement activation genes were elevated posttreatment, along with enrichment of tumor-associated macrophage signature. CONCLUSIONS: Durvalumab monotherapy conferred minimal efficacy in treating BCG-unresponsive CIS of the bladder, with 6-month CR of 12%. Complement activation is a potential mechanism behind treatment resistance.
Asunto(s)
Carcinoma in Situ , Carcinoma de Células Transicionales , Neoplasias de la Vejiga Urinaria , Humanos , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/patología , Vacuna BCG/efectos adversos , Vejiga Urinaria/patología , Anticuerpos Monoclonales/efectos adversos , Carcinoma in Situ/tratamiento farmacológico , Carcinoma in Situ/patología , Invasividad Neoplásica , Administración Intravesical , Adyuvantes Inmunológicos/uso terapéutico , Recurrencia Local de NeoplasiaRESUMEN
DNA can act as a scaffold for the cooperative binding of protein oligomers. For example, the phage 186 CI repressor forms a wheel of seven dimers wrapped in DNA with specific binding sites, while phage λ CI repressor dimers bind to two well-separated sets of operators, forming a DNA loop. Atomic force microscopy was used to measure transcription elongation by Escherichia coli RNA polymerase (RNAP) through these protein complexes. 186 CI, or λ CI, bound along unlooped DNA negligibly interfered with transcription by RNAP. Wrapped and looped topologies induced by these scaffolded, cooperatively bound repressor oligomers did not form significantly better roadblocks to transcription. Thus, despite binding with high affinity, these repressors are not effective roadblocks to transcription.
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Bacteriófago lambda , ARN Polimerasas Dirigidas por ADN , Sitios de Unión , ADN , Escherichia coli , Proteínas Reguladoras y Accesorias ViralesRESUMEN
Amyloid-ß peptides (Aß) assemble into both rigid amyloid fibrils and metastable oligomers termed AßO or protofibrils. In Alzheimer's disease, Aß fibrils constitute the core of senile plaques, but Aß protofibrils may represent the main toxic species. Aß protofibrils accumulate at the exterior of senile plaques, yet the protofibril-fibril interplay is not well understood. Applying chemical kinetics and atomic force microscopy to the assembly of Aß and lysozyme, protofibrils are observed to bind to the lateral surfaces of amyloid fibrils. When utilizing Aß variants with different critical oligomer concentrations, the interaction inhibits the autocatalytic proliferation of amyloid fibrils by secondary nucleation on the fibril surface. Thus, metastable oligomers antagonize their replacement by amyloid fibrils both by competing for monomers and blocking secondary nucleation sites. The protofibril-fibril interaction governs their temporal evolution and potential to exert specific toxic activities.
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Péptidos beta-Amiloides/metabolismo , Amiloide/metabolismo , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/genética , Cinética , Microscopía de Fuerza Atómica , Muramidasa/metabolismo , Agregado de Proteínas/fisiología , Unión Proteica , Propiedades de SuperficieRESUMEN
Assembly of amyloid fibrils and small globular oligomers is associated with a significant number of human disorders that include Alzheimer's disease, senile systemic amyloidosis, and type II diabetes. Recent findings implicate small amyloid oligomers as the dominant aggregate species mediating the toxic effects in these disorders. However, validation of this hypothesis has been hampered by the dearth of experimental techniques to detect, quantify, and discriminate oligomeric intermediates from late-stage fibrils, in vitro and in vivo. We have shown that the onset of significant oligomer formation is associated with a transition in thioflavin T kinetics from sigmoidal to biphasic kinetics. Here we showed that this transition can be exploited for screening fluorophores for preferential responses to oligomer over fibril formation. This assay identified crystal violet as a strongly selective oligomer-indicator dye for lysozyme. Simultaneous recordings of amyloid kinetics with thioflavin T and crystal violet enabled us to separate the combined signals into their underlying oligomeric and fibrillar components. We provided further evidence that this screening assay could be extended to amyloid-ß peptides under physiological conditions. Identification of oligomer-selective dyes not only holds the promise of biomedical applications but provides new approaches for unraveling the mechanisms underlying oligomer versus fibril formation in amyloid assembly.