23 research outputs found
Quantification of pharmacokinetic profiles of PD-1/PD-L1 antibodies by validated ELISAs
Immunotherapy has changed the paradigm of cancer treatments. In this way, several
combinatorial strategies based on monoclonal antibodies (mAb) such as anti (a)-PD-1 or anti (a)-PD-L1
are often reported to yield promising clinical benefits. However, the pharmacokinetic (PK) behavior
of these mAbs is a critical issue that requires selective analytical techniques. Indeed, few publications
report data on a-PD1/a-PD-L1 exposure and its relationship with therapeutic or toxic effects. In this
regard, preclinical assays allow the time profiles of antibody plasma concentrations to be characterized
rapidly and easily, which may help to increase PK knowledge. In this study, we have developed
and validated two in-house ELISAs to quantify a-PD-1 and a-PD-L1 in plasma collected from
tumor-bearing mice. The linear range for the a-PD-1 assay was 2.5–125 ng/mL and 0.11–3.125 ng/mL
for the a-PD-L1 assay, whereas the intra-and inter-day precision was lower than 20% for both analytes.
The PK characterization revealed a significant decrease in drug exposure after administration of
multiple doses. Plasma half-life for a-PD-1 was slightly shorter (22.3 h) than for a-PD-L1 (46.7 h).
To our knowledge, this is the first reported preclinical ELISA for these immune checkpoint inhibitors,
which is sufficiently robust to be used in different preclinical models. These methods can help to
understand the PK behavior of these antibodies under different scenarios and the relationship with
response, thus guiding the choice of optimal doses in clinical settings
Long-Term Systemic Expression of a Novel PD-1 Blocking Nanobody from an AAV Vector Provides Antitumor Activity without Toxicity
Immune checkpoint blockade using monoclonal antibodies (mAbs) able to block
programmed death-1 (PD-1)/PD-L1 axis represents a promising treatment for cancer. However,
it requires repetitive systemic administration of high mAbs doses, often leading to adverse effects.
We generated a novel nanobody against PD-1 (Nb11) able to block PD-1/PD-L1 interaction for
both mouse and human molecules. Nb11 was cloned into an adeno-associated virus (AAV) vector
downstream of four different promoters (CMV, CAG, EF1α, and SFFV) and its expression was
analyzed in cells from rodent (BHK) and human origin (Huh-7). Nb11 was expressed at high levels
in vitro reaching 2–20 micrograms/mL with all promoters, except SFFV, which showed lower levels.
Nb11 in vivo expression was evaluated in C57BL/6 mice after intravenous administration of AAV8
vectors. Nb11 serum levels increased steadily along time, reaching 1–3 microgram/mL two months
post-treatment with the vector having the CAG promoter (AAV-CAG-Nb11), without evidence of
toxicity. To test the antitumor potential of this vector, mice that received AAV-CAG-Nb11, or saline
as control, were challenged with colon adenocarcinoma cells (MC38). AAV-CAG-Nb11 treatment
prevented tumor formation in 30% of mice, significantly increasing survival. These data suggest that
continuous expression of immunomodulatory nanobodies from long-term expression vectors could
have antitumor effects with low toxicity
Local delivery of optimized nanobodies targeting the PD-1/PD-L1 axis with a self-amplifying RNA viral vector induces potent antitumor responses
Despite the success of immune checkpoint blockade for cancer therapy, many patients do not respond
adequately. We aimed to improve this therapy by optimizing both the antibodies and their delivery route, using
small monodomain antibodies (nanobodies) delivered locally with a self-amplifying RNA (saRNA) vector based
on Semliki Forest virus (SFV). We generated nanobodies against PD-1 and PD-L1 able to inhibit both human and
mouse interactions. Incorporation of a dimerization domain reduced PD-1/PD-L1 IC50 by 8- and 40-fold for antiPD-L1 and anti-PD-1 nanobodies, respectively. SFV viral particles expressing dimeric nanobodies showed a
potent antitumor response in the MC38 model, resulting in >50% complete regressions, and showed better
therapeutic efficacy compared to vectors expressing conventional antibodies. These effects were also observed in
the B16 melanoma model. Although a short-term expression of nanobodies was observed due to the cytopathic
nature of the saRNA vector, it was enough to generate a strong proinflammatory response in tumors, increasing
infiltration of NK and CD8+ T cells. Delivery of the SFV vector expressing dimeric nanobodies by local plasmid
electroporation, which could be more easily translated to the clinic, also showed a potent antitumor effect
Methyl donor supplementation in rats reverses the deleterious effect of maternal separation on depression-like behaviour
ZEB1/miR-200c/AGR2: A New Regulatory Loop Modulating the Epithelial-Mesenchymal Transition in Lung Adenocarcinomas
Epithelial-mesenchymal transition (EMT) is a process involved not only in morphogenesis
and embryonic development, but also in cancer progression, whereby tumor cells obtain a more
aggressive metastatic phenotype. Anterior gradient protein 2 (AGR2) maintains the epithelial
phenotype and blocks the induction of EMT, thus playing an undeniable role in tumor progression.
However, the mechanism through which AGR2 expression is regulated, not only during EMT, but
also in the early stages of cancer development, remains to be elucidated. In the present study, we
show an inverse correlation of AGR2 with ZEB1 (zinc finger enhancer binding protein, EF1) that
was verified by analysis of several independent clinical data sets of lung adenocarcinomas. We
also identified the ZEB1 binding site within the AGR2 promoter region and confirmed AGR2 as a
novel molecular target of ZEB1. The overexpression of ZEB1 decreased the promoter activity of the
AGR2 gene, which resulted in reduced AGR2 protein level and the acquisition of a more invasive
phenotype of these lung cancer cells. Conversely, silencing of ZEB1 led not only to increased levels of
AGR2 protein, but also attenuated the invasiveness of tumor cells. The AGR2 knockout, vice versa,
increased ZEB1 expression, indicating that the ZEB1/AGR2 regulatory axis may function in a double
negative feedback loop. In conclusion, we revealed for the first time that ZEB1 regulates AGR2 at
the transcriptional level, while AGR2 presence contributes to ZEB1 mRNA degradation. Thus, our
data identify a new regulatory mechanism between AGR2 and ZEB1, two rivals in the EMT process,
tightly associated with the development of metastasis
Postnatal maternal separation modifies the response to an obesogenic diet in adulthood in rats
SUMMARY
An early-life adverse environment has been implicated in the susceptibility to different diseases in adulthood, such as mental disorders, diabetes and obesity. We analyzed the effects of a high-fat sucrose (HFS) diet for 35 days in adult female rats that had experienced 180 minutes daily of maternal separation (MS) during lactancy. Changes in the obesity phenotype, biochemical profile, levels of glucocorticoid metabolism biomarkers, and the expression of different obesity- and glucocorticoid-metabolism-related genes were analyzed in periovaric adipose tissue. HFS intake increased body weight, adiposity and serum leptin levels, whereas MS decreased fat pad masses but only in rats fed an HFS diet. MS reduced insulin resistance markers but only in chow-fed rats. Corticosterone and estradiol serum levels did not change in this experimental model. A multiple gene expression analysis revealed that the expression of adiponutrin (Adpn) was increased owing to MS, and an interaction between HFS diet intake and MS was observed in the mRNA levels of leptin (Lep) and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (Ppargc1a). These results revealed that early-life stress affects the response to an HFS diet later in life, and that this response can lead to phenotype and transcriptomic changes
Adrenomedullin contributes to age-related memory loss in mice and is elevated in aging human brains
Quantification of pharmacokinetic profiles of PD-1/PD-L1 antibodies by validated ELISAs
Immunotherapy has changed the paradigm of cancer treatments. In this way, several
combinatorial strategies based on monoclonal antibodies (mAb) such as anti (a)-PD-1 or anti (a)-PD-L1
are often reported to yield promising clinical benefits. However, the pharmacokinetic (PK) behavior
of these mAbs is a critical issue that requires selective analytical techniques. Indeed, few publications
report data on a-PD1/a-PD-L1 exposure and its relationship with therapeutic or toxic effects. In this
regard, preclinical assays allow the time profiles of antibody plasma concentrations to be characterized
rapidly and easily, which may help to increase PK knowledge. In this study, we have developed
and validated two in-house ELISAs to quantify a-PD-1 and a-PD-L1 in plasma collected from
tumor-bearing mice. The linear range for the a-PD-1 assay was 2.5–125 ng/mL and 0.11–3.125 ng/mL
for the a-PD-L1 assay, whereas the intra-and inter-day precision was lower than 20% for both analytes.
The PK characterization revealed a significant decrease in drug exposure after administration of
multiple doses. Plasma half-life for a-PD-1 was slightly shorter (22.3 h) than for a-PD-L1 (46.7 h).
To our knowledge, this is the first reported preclinical ELISA for these immune checkpoint inhibitors,
which is sufficiently robust to be used in different preclinical models. These methods can help to
understand the PK behavior of these antibodies under different scenarios and the relationship with
response, thus guiding the choice of optimal doses in clinical settings
Long-Term Systemic Expression of a Novel PD-1 Blocking Nanobody from an AAV Vector Provides Antitumor Activity without Toxicity
Immune checkpoint blockade using monoclonal antibodies (mAbs) able to block programmed death-1 (PD-1)/PD-L1 axis represents a promising treatment for cancer. However, it requires repetitive systemic administration of high mAbs doses, often leading to adverse effects. We generated a novel nanobody against PD-1 (Nb11) able to block PD-1/PD-L1 interaction for both mouse and human molecules. Nb11 was cloned into an adeno-associated virus (AAV) vector downstream of four different promoters (CMV, CAG, EF1α, and SFFV) and its expression was analyzed in cells from rodent (BHK) and human origin (Huh-7). Nb11 was expressed at high levels in vitro reaching 2–20 micrograms/mL with all promoters, except SFFV, which showed lower levels. Nb11 in vivo expression was evaluated in C57BL/6 mice after intravenous administration of AAV8 vectors. Nb11 serum levels increased steadily along time, reaching 1–3 microgram/mL two months post-treatment with the vector having the CAG promoter (AAV-CAG-Nb11), without evidence of toxicity. To test the antitumor potential of this vector, mice that received AAV-CAG-Nb11, or saline as control, were challenged with colon adenocarcinoma cells (MC38). AAV-CAG-Nb11 treatment prevented tumor formation in 30% of mice, significantly increasing survival. These data suggest that continuous expression of immunomodulatory nanobodies from long-term expression vectors could have antitumor effects with low toxicity
JNK Activation in Alzheimer's Disease Is Driven by Amyloid β and Is Associated with Tau Pathology
c-Jun N-terminal kinase 3 (JNK3) is suggested to play a key role in neurodegenerative disorders, especially in Alzheimer's disease (AD). However, it remains unclear whether JNK or amyloid β (Aβ) appears first in the disease onset. Postmortem brain tissues from four dementia subtypes of patients (frontotemporal dementia, Lewy body dementia, vascular dementia, and AD) were used to measure activated JNK (pJNK) and Aβ levels. pJNK expression is significantly increased in AD; however, similar pJNK expression was found in other dementias. Furthermore, there was a significant correlation, co-localization, and direct interaction between pJNK expression and Aβ levels in AD. Significant increased levels of pJNK were also found in Tg2576 mice, a model of AD. In this line, Aβ42 intracerebroventricular injection in wild-type mice was able to induce a significant elevation of pJNK levels. JNK3 overexpression, achieved by intrahippocampal injection of an adeno-associated viral vector expressing this protein, was enough to induce cognitive deficiencies and precipitate Tau aberrant misfolding in Tg2576 mice without accelerating amyloid pathology. JNK3 overexpression may therefore be triggered by increased Aβ. The latter, together with subsequent involvement of Tau pathology, may be underlying cognitive alterations in early stages of AD