Biology subjects

Hussack, G.

Publications and source records attributed to Hussack, G..

4 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↗

bioRxiv2023

HSV-1 Oncolytic Virus Targeting CEACAM6-Expressing Tumors Using a Bispecific T-Cell Engager

VG21306 is a novel oncolytic virus (OV) that encodes a secretable bispecific T-cell engager targeting Carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6)-expressing tumors. Delivering a T-cell engager locally to a tumor mass will circumvent physical barriers that prevent antibodies from penetrating the tumor, and will mitigate the off-tumor, on-target toxicity risk. Both in vitro and in vivo testing demonstrated the expression of a functional T-cell engager capable of binding both targets. The efficacy of the engager was demonstrated in vitro, where addition of the engager payload to the OV enhanced anti-tumor efficacy against tumor cells overexpressing CEACAM6. Moreover, we have demonstrated the engagers ability to induce bystander killing in cells lacking CEACAM6 expression, as well as engaging exhausted T cells and inducing tumor cell death. The safety of the engager was demonstrated by the lack of binding to normal human tissue or normal tissue adjacent to tumors, as well as the absence of any measurable leakage of the expressed engager into the blood of mice treated by intratumoral OV injection.

cancer biology↗

bioRxiv2021

Arsenal of Nanobodies for Broad-Spectrum Countermeasures against Current and Future SARS-CoV-2 Variants of Concerns

Nanobodies offer several potential advantages over mAbs for the control of SARS-CoV-2. Their ability to access cryptic epitopes conserved across SARS-CoV-2 variants of concern (VoCs) and feasibility to engineer modular, multimeric designs, make these antibody fragments ideal candidates for developing broad-spectrum therapeutics against current and continually emerging SARS-CoV-2 VoCs. Here we describe a diverse collection of 37 anti-SARS-CoV-2 spike glycoprotein nanobodies extensively characterized as both monovalent and IgG Fc-fused bivalent modalities. The panel of nanobodies were shown to have high intrinsic affinity; high thermal, thermodynamic and aerosolization stability; broad subunit/domain specificity and cross-reactivity across many VoCs; wide-ranging epitopic and mechanistic diversity; high and broad in vitro neutralization potencies; and high neutralization efficacies in hamster models of SARS-CoV-2 infection, reducing viral burden by up to six orders of magnitude to below detectable levels. In vivo protection was demonstrated with anti-RBD and previously unreported anti-NTD and anti-S2 nanobodies. This collection of nanobodies provides a therapeutic toolbox from which various cocktails or multi-paratopic formats could be built to tackle current and future SARS-CoV-2 variants and SARS-related viruses. Furthermore, the high aerosol-ability of nanobodies provides the option for effective needle-free delivery through inhalation.

bioengineering↗

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↗