Engineering a natural ligand-based CAR: directed evolution of the stress-receptor NKp30
| Autor: | Margaret E. Ackerman, Savannah E. Butler, Cheryl H. Chang, Yina H. Huang, Charles L. Sentman, Rachel A. Brog |
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| Rok vydání: | 2021 |
| Předmět: |
Cancer Research
B7 Antigens CD3 Complex Protein Conformation Immunology Cell Separation Yeast display Ligands Article 03 medical and health sciences Mice 0302 clinical medicine CD28 Antigens Cell Line Tumor Immunology and Allergy Animals Humans Receptor Gene Library Natural Cytotoxicity Triggering Receptor 3 Receptors Chimeric Antigen Chemistry Gene Expression Profiling CD28 Genetic Variation Ligand (biochemistry) Directed evolution Flow Cytometry Chimeric antigen receptor Cell biology Kinetics HEK293 Cells Oncology Mutation Cytokines Immunotherapy Intracellular Function (biology) 030215 immunology Single-Chain Antibodies |
| Zdroj: | Cancer Immunol Immunother |
| ISSN: | 1432-0851 |
| Popis: | B7H6, a stress-induced ligand which binds to the NK cell receptor NKp30, has recently emerged as a promising candidate for immunotherapy due to its tumor-specific expression on a broad array of human tumors. NKp30 can function as a chimeric antigen receptor (CAR) extracellular domain but exhibits weak binding with a fast on and off rate to B7H6 compared to the TZ47 anti-B7H6 single-chain variable fragment (scFv). Here, directed evolution using yeast display was employed to isolate novel NKp30 variants that bind to B7H6 with higher affinity compared to the native receptor but retain its fast association and dissociation profile. Two variants, CC3 and CC5, were selected for further characterization and were expressed as soluble Fc-fusion proteins and CARs containing CD28 and CD3ς intracellular domains. We observed that Fc-fusion protein forms of NKp30 and its variants were better able to bind tumor cells expressing low levels of B7H6 than TZ47, and that the novel variants generally exhibited improved in vitro tumor cell killing relative to NKp30. Interestingly, CAR T cells expressing the engineered variants produced unique cytokine signatures in response to multiple tumor types expressing B7H6 compared to both NKp30 and TZ47. These findings suggest that natural CAR receptors can be fine-tuned to produce more desirable signaling outputs while maintaining evolutionary advantages in ligand recognition relative to scFvs. |
| Databáze: | OpenAIRE |
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