Biology subjects

Raphael, S.

Publications and source records attributed to Raphael, S..

3 recordsLinked to original sources

bioRxiv2024

Broadly Reactive Anti-VHH Antibodies for Characterizing, Blocking, or Activating Nanobody-Based CAR-T Cells

Production of chimeric antigen receptor T cell (CAR-T) therapies is dependent on the use of antibody reagents to label, isolate, and/or expand T cell products. We sought to create antibody-based tools that directly target the variable domain of heavy-chain only antibodies (VHH or nanobody) used in some CAR molecules. Two murine antibodies were identified which bind to distinct epitopes in the conserved framework regions of llama-derived VHHs, and not to human VH domains. We produced a high-quality dual-clonal anti-VHH antibody product which reacts with over 98% of VHH proteins, regardless of their antigenic specificity. Anti-VHH binding did not disrupt VHH/antigen interaction, and thus could be used for secondary labeling to assess cellular or tissue reactivity of VHH molecules. Despite not interfering with antigen binding, anti-VHH antibodies potently inhibited VHH-CAR function, blocking CAR-T activation and cytolytic killing of target cells. When immobilized, anti-VHH antibodies could also be applied for activation and expansion of VHH CAR-T cells, inducing 730-fold mean expansion, >94% CAR purity, with retained CD8/CD4 heterogeneity. Functionally, anti-VHH antibody-expanded CAR-T cells maintained strong antigen specific activity without functional exhaustion. Overall, these data identify a useful new tool for understanding and manipulating VHH-based CAR-T cells. Funding SourceThis work was funded by the National Research Council Canada Disruptive Technology Solutions Cell and Gene Therapy challenge program, and BioCanRx Declaration of interestsThe anti-VHH antibodies reported here are the subject of a provisional patent application by the National Research Council of Canada

immunology↗

bioRxiv2018

Single-domain antibodies represent novel alternatives to monoclonal antibodies as targeting agents against the human papillomavirus 16 E6 protein

Approximately one-fifth of all malignancies worldwide are etiologically-associated with a persistent viral or bacterial infection. Thus, there is particular interest in therapeutic molecules which utilize components of a natural immune response to specifically inhibit oncogenic microbial proteins, as it is anticipated they will elicit fewer off-target effects than conventional treatments. This concept has been explored in the context of human papillomavirus type 16 (HPV16)-related cancers, through the development of monoclonal antibodies and fragments thereof against the viral E6 oncoprotein. However, challenges related to the biology of E6 as well as the functional properties of the antibodies themselves appear to have precluded their clinical translation. In this study, we attempted to address these issues by exploring the utility of the variable domains of camelid heavy-chain-only antibodies (denoted as VHHs). Through the construction and panning of two llama immune VHH phage display libraries, a pool of potential VHHs was isolated. The interactions of these VHHs with recombinant E6 protein were further characterized using ELISA, Western blotting under both denaturing and native conditions, as well as surface plasmon resonance, and three antibodies were identified that bound recombinant E6 with affinities in the nanomolar range. Our results now lead the way for subsequent studies into the ability of these novel molecules to inhibit HPV16-infected cells in vitro and in vivo.

cancer biology↗

bioRxiv2018

Studies of NH4+ and NO3- uptake ability of subalpine plants and resource-use strategy identified by their functional traits.

AO_SCPLOWBSTRACTC_SCPLOWThe leaf economics spectrum (LES) is based on a suite of leaf traits related to plant functioning and ranges from resource-conservative to resource-acquisitive strategies. However, the relationships with root traits, and the associated belowground plant functioning such as N uptake, including nitrate (NO3-) and ammonium (NH4+), is still poorly known. Additionally, environmental variations occurring both in time and in space could uncouple LES from root traits. We explored, in subalpine grasslands, the relationships between leaf and root morphological traits for 3 dominant perennial grass species, and to what extent they contribute to the whole-plant economics spectrum. We also investigated the link between this spectrum and NO3- and NH4+ uptake rates, as well as the variations of uptake across four grasslands differing by the land-use history at peak biomass and in autumn. Although poorly correlated with leaf traits, root traits contributed to an economic spectrum at the whole plant level. Higher NH4+ and NO3- uptake abilities were associated with the resource-acquisitive strategy. Nonetheless, NH4+ and NO3- uptake within species varied between land-uses and with sampling time, suggesting that LES and plant traits are good, but still incomplete, descriptors of plant functioning. Although the NH4+: NO3- uptake ratio was different between plant species in our study, they all showed a preference for NH4+, and particularly the most conservative species. Soil environmental variations between grasslands and sampling times may also drive to some extent the NH4+ and NO3- uptake ability of species. Our results support the current efforts to build a more general framework including above- and below-ground processes when studying plant community functioning.

ecology↗