Discovery and preclinical development of a therapeutically active nanobody-based chimeric antigen receptor targeting human CD22

Scott McComb; Mehdi Arbabi-Ghahroudi; Kevin A Hay; Brian A Keller; Sharlene Faulkes; Michael Rutherford; Tina Nguyen; Alex Shepherd; Cunle Wu; Anne Marcil; Annie Aubry; Greg Hussack; Devanand M Pinto; Shannon Ryan; Shalini Raphael; Henk van Faassen; Ahmed Zafer; Qin Zhu; Susanne Maclean; Anindita Chattopadhyay; Komal Gurnani; Rénald Gilbert; Christine Gadoury; Umar Iqbal; Dorothy Fatehi; Anna Jezierski; Jez Huang; Robert A Pon; Mhairi Sigrist; Robert A Holt; Brad H Nelson; Harold Atkins; Natasha Kekre; Eric Yung; John Webb; Julie S Nielsen; Risini D Weeratna

doi:10.1016/j.omton.2024.200775

Discovery and preclinical development of a therapeutically active nanobody-based chimeric antigen receptor targeting human CD22

Mol Ther Oncol. 2024 Feb 13;32(1):200775. doi: 10.1016/j.omton.2024.200775. eCollection 2024 Mar 21.

Authors

Scott McComb^{1

2

3}, Mehdi Arbabi-Ghahroudi^{1

2}, Kevin A Hay^{4

5}, Brian A Keller^{6

7}, Sharlene Faulkes⁸, Michael Rutherford^{6

9}, Tina Nguyen¹, Alex Shepherd^{1

2}, Cunle Wu^{1

10}, Anne Marcil¹, Annie Aubry¹, Greg Hussack¹, Devanand M Pinto¹, Shannon Ryan¹, Shalini Raphael¹, Henk van Faassen¹, Ahmed Zafer¹, Qin Zhu¹, Susanne Maclean¹, Anindita Chattopadhyay¹, Komal Gurnani¹, Rénald Gilbert¹, Christine Gadoury¹, Umar Iqbal¹, Dorothy Fatehi¹, Anna Jezierski^{1

2}, Jez Huang¹, Robert A Pon¹, Mhairi Sigrist⁴, Robert A Holt^{11

12

13}, Brad H Nelson^{14

11}, Harold Atkins¹⁵, Natasha Kekre^{15

16}, Eric Yung¹², John Webb¹⁴, Julie S Nielsen¹⁴, Risini D Weeratna¹

Affiliations

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.

Abstract

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.

Keywords: CAR optimization; CAR-T; CD22; MT: Regular Issue; cell therapy; chimeric antigen receptors; hematological malignancy; leukemia and lymphoma; nanobody; preclinical development; single-domain antibody.