Long-Term Systemic Expression of a Novel PD-1 Blocking Nanobody from an AAV Vector Provides Antitumor Activity
Noelia Silva-Pilipich1,2,3, Eva Martisova1,2, María Cristina Ballesteros-Briones1,2
1Division of Gene Therapy and Regulation of Gene Expression, Cima Universidad de Navarra, 31008 Pamplona, Spain.
Abstract:
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.
Insights
A novel nanobody targeting programmed death-1 (PD-1) delivered via adeno-associated virus (AAV) vectors shows potential for cancer treatment. This approach demonstrated antitumor effects and increased survival in mice with reduced toxicity compared to traditional antibody therapies.
Area of Science:
- Immunology
- Gene Therapy
- Oncology
Background:
- Immune checkpoint blockade using monoclonal antibodies (mAbs) targeting the programmed death-1 (PD-1)/PD-L1 axis is a promising cancer therapy.
- Current mAb treatments require frequent systemic administration, leading to potential adverse effects.
Purpose of the Study:
- To develop and evaluate a novel nanobody (Nb11) against PD-1 for blocking PD-1/PD-L1 interactions.
- To assess the efficacy and safety of expressing Nb11 using adeno-associated virus (AAV) vectors for sustained therapeutic effect.
Main Methods:
- A nanobody (Nb11) targeting PD-1 was engineered and cloned into AAV vectors with different promoters (CMV, CAG, EF1α, SFFV).
- In vitro expression was analyzed in rodent (BHK) and human (Huh-7) cells.
- In vivo expression and toxicity were evaluated in C57BL/6 mice following intravenous AAV8 administration.
- Antitumor efficacy was tested in MC38 colon adenocarcinoma mouse models.
Main Results:
- Nb11 was expressed at high levels in vitro and in vivo, with sustained serum levels up to 1-3 µg/mL two months post-treatment using the AAV-CAG-Nb11 vector.
- No evidence of toxicity was observed in mice treated with AAV-CAG-Nb11.
- AAV-CAG-Nb11 treatment prevented tumor formation in 30% of mice and significantly increased survival.
Conclusions:
- Continuous expression of immunomodulatory nanobodies via long-term expression vectors like AAV offers a potential strategy for cancer therapy.
- This gene therapy approach demonstrates promising antitumor effects with a favorable safety profile.
- Nb11 delivered via AAV represents a novel alternative to conventional antibody-based immunotherapies.
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