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

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.

SUBMITTER: McComb S

PROVIDER: S-EPMC10914482 | biostudies-literature | 2024 Mar

REPOSITORIES: biostudies-literature

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

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

Molecular therapy. Oncology 20240213 1

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. Immunizat ...[more]

PMID: 38596311

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Project description:Rationale: CD39, a key ectonucleotidase that drives adenosine production, acts as a critical immunosuppressive checkpoint in cancer. Although it has shown promise as a therapeutic target, clinical trials are demonstrating the need for more potent targeting approaches. This need is driving innovation towards the development of novel antibodies and the exploration of strategic combinations with a range of immunotherapies. Methods: An anti-CD39 nanobody was screened and tested for its affinity and binding ability using biolayer interferometry, ELISA and flow cytometry. Blocking ability against soluble and membrane-bound CD39 was measured after CD39 blockade. Internalization was detected using immunofluorescence. The reversal of T-cell function by the anti-CD39 antibody was assessed by CFSE-based T-cell proliferation, CD25 expression and IFN-γ secretion. The in vivo function of tumor growth inhibition was further tested in a mouse model and we also tested the phenotype of immune cells after CD39 antibody administration from tumor tissue, draining lymph nodes and peripheral blood. We inserted the antibody sequence into the chimeric antigen receptor (CAR) construct to induce MSLN CAR-T cells to secret the CD39 antibody, and the efficacy was measured in xenograft models of ovarian cancer. Results: We screened human CD39 antibodies using a VHH library and developed a single-epitope anti-CD39 nanobody, named huCD39 mAb, with high affinity and potent binding and blocking ability. The huCD39 mAb was internalized in a time-dependent manner. The in vitro study revealed that the huCD39 mAb was highly effective in enhancing T-cell proliferation and functionality. In vivo, the huCD39 mAb showed significant anti-tumor efficacy in an immunocompetent mouse model. Flow cytometry analysis demonstrated downregulated CD39 expression in immune cells after antibody administration. We also observed increased CD39 expression in ovarian cancer tissue and in activated CAR T cells. Subsequently, we developed a type of MSLN CAR-T cells secreting huCD39 mAb which showed effective eradication or inhibition in ovarian tumor xenografts. Conclusions: A novel huCD39 mAb with strong blocking ability against human CD39 and potent inhibition of tumor growth has been developed. Furthermore, a modified huCD39 mAb-secreting CAR-T cell has been generated, exhibiting superior efficacy against ovarian cancer. This provides a promising strategy for optimizing immunotherapies in ovarian cancer and potentially other malignancies.

Dataset Information

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

Publications

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

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