title: Versatile strategy for controlling the specificity and activity of engineered T cells.
abstract: The adoptive transfer of autologous T cells engineered to express a chimeric antigen receptor (CAR) has emerged as a promising cancer therapy. Despite impressive clinical efficacy, the general application of current CAR-T--cell therapy is limited by serious treatment-related toxicities. One approach to improve the safety of CAR-T cells involves making their activation and proliferation dependent upon adaptor molecules that mediate formation of the immunological synapse between the target cancer cell and T-cell. Here, we describe the design and synthesis of structurally defined semisynthetic adaptors we refer to as "switch" molecules, in which anti-CD19 and anti-CD22 antibody fragments are site-specifically modified with FITC using genetically encoded noncanonical amino acids. This approach allows the precise control over the geometry and stoichiometry of complex formation between CD19- or CD22-expressing cancer cells and a "universal" anti-FITC-directed CAR-T cell. Optimization of this CAR-switch combination results in potent, dose-dependent in vivo antitumor activity in xenograft models. The advantage of being able to titrate CAR-T-cell in vivo activity was further evidenced by reduced in vivo toxicity and the elimination of persistent B-cell aplasia in immune-competent mice. The ability to control CAR-T cell and cancer cell interactions using intermediate switch molecules may expand the scope of engineered T-cell therapy to solid tumors, as well as indications beyond cancer therapy.
authors.affiliation: Department of Biology, California Institute for Biomedical Research, La Jolla, CA 92037;
authors.forename: Jennifer S Y
authors.initials: JS
authors.lastname: Ma
authors.affiliation: Department of Biology, California Institute for Biomedical Research, La Jolla, CA 92037;
authors.forename: Ji Young
authors.initials: JY
authors.lastname: Kim
authors.affiliation: Department of Biology, California Institute for Biomedical Research, La Jolla, CA 92037;
authors.forename: Stephanie A
authors.initials: SA
authors.lastname: Kazane
authors.affiliation: Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037;
authors.forename: Sei-Hyun
authors.initials: SH
authors.lastname: Choi
authors.affiliation: Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037;
authors.forename: Hwa Young
authors.initials: HY
authors.lastname: Yun
authors.affiliation: Department of Biology, California Institute for Biomedical Research, La Jolla, CA 92037;
authors.forename: Min Soo
authors.initials: MS
authors.lastname: Kim
authors.affiliation: Department of Biology, California Institute for Biomedical Research, La Jolla, CA 92037;
authors.forename: David T
authors.initials: DT
authors.lastname: Rodgers
authors.affiliation: Department of Biology, California Institute for Biomedical Research, La Jolla, CA 92037;
authors.forename: Holly M
authors.initials: HM
authors.lastname: Pugh
authors.affiliation: Department of Biology, California Institute for Biomedical Research, La Jolla, CA 92037;
authors.forename: Oded
authors.initials: O
authors.lastname: Singer
authors.affiliation: Department of Biology, California Institute for Biomedical Research, La Jolla, CA 92037;
authors.forename: Sophie B
authors.initials: SB
authors.lastname: Sun
authors.affiliation: Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA 92037; SCIEX Separations, Brea, CA 92821;
authors.forename: Bryan R
authors.initials: BR
authors.lastname: Fonslow
authors.affiliation: Experimental Transplantation and Immunology Branch, National Institutes of Health, National Cancer Institute, Bethesda, MD 20892.
authors.forename: James N
authors.initials: JN
authors.lastname: Kochenderfer
authors.affiliation: Department of Biology, California Institute for Biomedical Research, La Jolla, CA 92037;
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Wright
authors.affiliation: Department of Biology, California Institute for Biomedical Research, La Jolla, CA 92037; Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037; schultz@scripps.edu tyoung@calibr.org chkim@calibr.org.
authors.forename: Peter G
authors.initials: PG
authors.lastname: Schultz
authors.affiliation: Department of Biology, California Institute for Biomedical Research, La Jolla, CA 92037; schultz@scripps.edu tyoung@calibr.org chkim@calibr.org.
authors.forename: Travis S
authors.initials: TS
authors.lastname: Young
authors.affiliation: Department of Biology, California Institute for Biomedical Research, La Jolla, CA 92037; schultz@scripps.edu tyoung@calibr.org chkim@calibr.org.
authors.forename: Chan Hyuk
authors.initials: CH
authors.lastname: Kim
authors.affiliation: Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037;
authors.forename: Yu
authors.initials: Y
authors.lastname: Cao
mesh_terms: D000818:Animals; D018941:Antigens, CD19 / Q000276:immunology*; D000951:Antigens, Neoplasm / Q000276:immunology*; D001386:Azides; D001402:B-Lymphocytes / Q000276:immunology / Q000473:pathology; D045744:Cell Line, Tumor; D003602:Cytotoxicity, Immunologic; D005260:Female; D016650:Fluorescein-5-isothiocyanate; D005822:Genetic Vectors; D006801:Humans; D016219:Immunotherapy, Adoptive / Q000009:adverse effects / Q000379:methods*; D016086:Lentivirus / Q000235:genetics; D015448:Leukemia, B-Cell / Q000628:therapy*; D008213:Lymphocyte Activation; D008231:Lymphopenia / Q000209:etiology / Q000517:prevention & control; D051379:Mice; D008810:Mice, Inbred C57BL; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D008958:Models, Molecular; D010649:Phenylalanine / Q000031:analogs & derivatives; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D011948:Receptors, Antigen, T-Cell / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000276:immunology; D051918:Sialic Acid Binding Ig-like Lectin 2 / Q000276:immunology*; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology; D037182:T-Cell Antigen Receptor Specificity; D013601:T-Lymphocytes / Q000276:immunology* / Q000637:transplantation; D014161:Transduction, Genetic; D023041:Xenograft Model Antitumor Assays
chemical_list: D018941:Antigens, CD19; D000951:Antigens, Neoplasm; D001386:Azides; C497867:CD22 protein, human; C000598123:CTL019 chimeric antigen receptor; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; D051918:Sialic Acid Binding Ig-like Lectin 2; D057127:Single-Chain Antibodies; C027162:4-azidophenylalanine; D010649:Phenylalanine; D016650:Fluorescein-5-isothiocyanate
keywords: B-cell aplasia; cancer immunotherapy; chimeric antigen receptor T cell; cytokine release syndrome; noncanonical amino acids