1 Human Health Therapeutics Research Centre, National Research Council, Ottawa, ON, Canada.
2 Department of Biochemistry, Microbiology, and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada.
3 Centre for Infection, Immunity, and Inflammation, University of Ottawa, Ottawa, ON, Canada.
4 Terry Fox Laboratory, British Columbia Cancer Research Institute, Vancouver, BC, Canada.
5 Division of Hematology, Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada.
6 Division of Anatomical Pathology, The Ottawa Hospital/University of Ottawa, Ottawa, ON, Canada.
7 University of Ottawa Faculty of Medicine, Ottawa, ON, Canada.
8 Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada.
9 Division of Hematopathology and Transfusion Medicine, The Ottawa Hospital/University of Ottawa, Ottawa, ON, Canada.
10 Department of Biology, Concordia University, Montréal, QC, Canada.
11 Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada.
12 Canada's Michael Smith Genome Sciences Centre, Vancouver, BC, Canada.
13 Department of Molecular Biology & Biochemistry, Simon Fraser University, Burnaby, BC, Canada.
14 Deeley Research Centre, British Columbia Cancer Research Institute, Victoria, BC, Canada.
15 Division of Hematology, Department of Medicine, The Ottawa Hospital, Ottawa, ON, Canada.
16 Clinical Epidemiology Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada.

Chimeric antigen receptor (CAR) T cell therapies targeting B cell-restricted antigens CD19, CD20, or CD22 can produce potent clinical responses for some B cell malignancies, but relapse remains common. Camelid single-domain antibodies (sdAbs or nanobodies) are smaller, simpler, and easier to recombine than single-chain variable fragments (scFvs) used in most CARs, but fewer sdAb-CARs have been reported. Thus, we sought to identify a therapeutically active sdAb-CAR targeting human CD22. Immunization of an adult Llama glama with CD22 protein, sdAb-cDNA library construction, and phage panning yielded >20 sdAbs with diverse epitope and binding properties. Expressing CD22-sdAb-CAR in Jurkat cells drove varying CD22-specific reactivity not correlated with antibody affinity. Changing CD28- to CD8-transmembrane design increased CAR persistence and expression in vitro. CD22-sdAb-CAR candidates showed similar CD22-dependent CAR-T expansion in vitro, although only membrane-proximal epitope targeting CD22-sdAb-CARs activated direct cytolytic killing and extended survival in a lymphoma xenograft model. Based on enhanced survival in blinded xenograft studies, a lead CD22sdCAR-T was selected, achieving comparable complete responses to a benchmark short linker m971-scFv CAR-T in high-dose experiments. Finally, immunohistochemistry and flow cytometry confirm tissue and cellular-level specificity of the lead CD22-sdAb. This presents a complete report on preclinical development of a novel CD22sdCAR therapeutic.