8 research outputs found
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
Short-term local expression of a PD-L1 blocking antibody from a self-replicating RNA vector induces potent antitumor responses
Immune checkpoint blockade has shown anti-cancer efficacy, but requires systemic administration of monoclonal antibodies (mAbs), often leading to adverse effects. To avoid toxicity, mAbs could be expressed locally in tumors. We developed adeno-associated virus (AAV) and Semliki Forest virus (SFV) vectors expressing anti-programmed death ligand 1 (aPDL1) mAb. When injected intratumorally in MC38 tumors, both viral vectors led to similar local mAb expression at 24 h, diminishing quickly in SFV-aPDL1-treated tumors. However, SFV-aPDL1 induced >40% complete regressions and was superior to AAV-aPDL1, as well as to aPDL1 mAb given systemically or locally. SFV-aPDL1 induced abscopal effects and was also efficacious against B16-ovalbumin (OVA). The higher SFV-aPDL1 antitumor activity could be related to local upregulation of interferon-stimulated genes because of SFV RNA replication. This was confirmed by combining local SFV-LacZ administration and systemic aPDL1 mAb, which provided higher antitumor effects than each separated agent. SFVaPDL1 promoted tumor-specific CD8 T cells infiltration in both tumor models. In MC38, SFV-aPDL1 upregulated co-stimulatory markers (CD137/OX40) in tumor CD8 T cells, and its combination with anti-CD137 mAb showed more pronounced antitumor effects than each single agent. These results indicate that local transient expression of immunomodulatory mAbs using non-propagative RNA vectors inducing type I interferon (IFN-I) responses represents a potent and
Optimization of a GDNF production method based on Semliki Forest virus vector
Human glial cell line-derived neurotrophic factor (hGDNF) is the most potent dopaminergic factor described so far, and it is therefore considered a promising drug for Parkinson’s disease (PD) treatment. However, the production of therapeutic proteins with a high degree of purity and a specific glycosylation pattern is a major
challenge that hinders its commercialization. Although a variety of systems can be used for protein production, only a small number of them are suitable to produce clinical-grade proteins. Specifically, the baby hamster kidney cell line (BHK-21) has shown to be an effective system for the expression of high levels of hGDNF, with appropriate post-translational modifications and protein folding. This system, which is based on the electroporation of BHK-21 cells using a Semliki Forest virus (SFV) as expression vector, induces a strong shut-off of host cell protein synthesis that simplify the purification process. However, SFV vector exhibits a temperature dependent cytopathic effect on host cells, which could limit hGDNF expression. The aim of this study was to improve the expression and purification of hGDNF using a biphasic temperature cultivation protocol that would decrease the cytopathic effect induced by SFV. Here we show that an increase in the temperature from 33◦C to 37◦C during the “shut-off period”, produced a significant improvement in cell survival and hGDNF expression. Inconsonance, this protocol led to the production of almost 3-fold more hGDNF when compared to the previously described methods. Therefore, a “recovery period” at 37◦C before cells are exposed at 33◦C is crucial to maintain cell viability and increase hGDNF expression. The protocol described constitutes an efficient and highly scalable method to produce highly pure hGDNF
Optimization of a GDNF production method based on Semliki Forest virus vector
Human glial cell line-derived neurotrophic factor (hGDNF) is the most potent dopaminergic factor described so far, and it is therefore considered a promising drug for Parkinson’s disease (PD) treatment. However, the production of therapeutic proteins with a high degree of purity and a specific glycosylation pattern is a major
challenge that hinders its commercialization. Although a variety of systems can be used for protein production, only a small number of them are suitable to produce clinical-grade proteins. Specifically, the baby hamster kidney cell line (BHK-21) has shown to be an effective system for the expression of high levels of hGDNF, with appropriate post-translational modifications and protein folding. This system, which is based on the electroporation of BHK-21 cells using a Semliki Forest virus (SFV) as expression vector, induces a strong shut-off of host cell protein synthesis that simplify the purification process. However, SFV vector exhibits a temperature dependent cytopathic effect on host cells, which could limit hGDNF expression. The aim of this study was to improve the expression and purification of hGDNF using a biphasic temperature cultivation protocol that would decrease the cytopathic effect induced by SFV. Here we show that an increase in the temperature from 33◦C to 37◦C during the “shut-off period”, produced a significant improvement in cell survival and hGDNF expression. Inconsonance, this protocol led to the production of almost 3-fold more hGDNF when compared to the previously described methods. Therefore, a “recovery period” at 37◦C before cells are exposed at 33◦C is crucial to maintain cell viability and increase hGDNF expression. The protocol described constitutes an efficient and highly scalable method to produce highly pure hGDNF
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
Short-term local expression of a PD-L1 blocking antibody from a self-replicating RNA vector induces potent antitumor responses
Immune checkpoint blockade has shown anti-cancer efficacy, but requires systemic administration of monoclonal antibodies (mAbs), often leading to adverse effects. To avoid toxicity, mAbs could be expressed locally in tumors. We developed adeno-associated virus (AAV) and Semliki Forest virus (SFV) vectors expressing anti-programmed death ligand 1 (aPDL1) mAb. When injected intratumorally in MC38 tumors, both viral vectors led to similar local mAb expression at 24 h, diminishing quickly in SFV-aPDL1-treated tumors. However, SFV-aPDL1 induced >40% complete regressions and was superior to AAV-aPDL1, as well as to aPDL1 mAb given systemically or locally. SFV-aPDL1 induced abscopal effects and was also efficacious against B16-ovalbumin (OVA). The higher SFV-aPDL1 antitumor activity could be related to local upregulation of interferon-stimulated genes because of SFV RNA replication. This was confirmed by combining local SFV-LacZ administration and systemic aPDL1 mAb, which provided higher antitumor effects than each separated agent. SFVaPDL1 promoted tumor-specific CD8 T cells infiltration in both tumor models. In MC38, SFV-aPDL1 upregulated co-stimulatory markers (CD137/OX40) in tumor CD8 T cells, and its combination with anti-CD137 mAb showed more pronounced antitumor effects than each single agent. These results indicate that local transient expression of immunomodulatory mAbs using non-propagative RNA vectors inducing type I interferon (IFN-I) responses represents a potent and