Figure 1.
Discovery and optimization of CD300a TASR. (A) Overview of allo–T-cell + NK challenge assay. Survival of HLA-I+ and B2M KO target T cells from an HLA-A2+ donor challenged with NK and allo-T-cell effectors from HLA-A2− donors. Allo-T cells express the AHIII.2 TCRαβ reactive to EMC7 peptide, ALWGFFPVL, presented by HLA-A2. To discern from allo-T cells, B2M KO target T cells were engineered to express RQR8 and HLA-I+ target T cells were engineered to express GFP. (B) HLA-I expression by flow cytometry of HLA-I+ and B2M KO target T cells from panel A. (C) NK challenge assay to screen cloaking strategies on B2M KO T cells. Ligands are expressed by mRNA electroporation (EP), and KO by CRISPR/Cas9. y-axis shows ratio of IC50 value of the indicated cloaked T cell over uncloaked negative control. IC50 represents E:T ratio corresponding to 50% target T-cell survival, derived from one 6-7-point curve of T-cell survival at various E:T ratios challenged with 1 NK cell donor, with higher values indicating enhanced persistence. N = 1 technical replicate per condition. (D) Annotation of TASR structure. (E-G) Effect of hinge length on function of CD300a TASR. (E) Design of CD300a TASR variants with different hinge domains with the indicated amino acid lengths and their expression level after mRNA EP into B2M KO T cells. (F) NK challenge assay expressing the indicated CD300a TASR variant from panel E. N = 2 technical replicate curves per condition. (G) Correlation of NK protection from panel F as defined by IC50 value with hinge length from panel E. Error bar indicates 95% confidence interval of IC50 value. (H-J) Final optimization of CD300a TASR. (H) CD300a TASR V1 and 2 optimized variants are tested for expression via mRNA EP of B2M KO T cells. (I) NK challenge assay against the indicated CD300a TASR variant from (H). N = 1 technical replicate curve per condition. (J) Optimized CD300a TASR design and mechanism of action. GFP, green fluorescent protein; IgG, immunoglobulin G; ITIM, immunoreceptor tyrosine–based inhibitory motif.

Discovery and optimization of CD300a TASR. (A) Overview of allo–T-cell + NK challenge assay. Survival of HLA-I+ and B2M KO target T cells from an HLA-A2+ donor challenged with NK and allo-T-cell effectors from HLA-A2 donors. Allo-T cells express the AHIII.2 TCRαβ reactive to EMC7 peptide, ALWGFFPVL, presented by HLA-A2. To discern from allo-T cells, B2M KO target T cells were engineered to express RQR8 and HLA-I+ target T cells were engineered to express GFP. (B) HLA-I expression by flow cytometry of HLA-I+ and B2M KO target T cells from panel A. (C) NK challenge assay to screen cloaking strategies on B2M KO T cells. Ligands are expressed by mRNA electroporation (EP), and KO by CRISPR/Cas9. y-axis shows ratio of IC50 value of the indicated cloaked T cell over uncloaked negative control. IC50 represents E:T ratio corresponding to 50% target T-cell survival, derived from one 6-7-point curve of T-cell survival at various E:T ratios challenged with 1 NK cell donor, with higher values indicating enhanced persistence. N = 1 technical replicate per condition. (D) Annotation of TASR structure. (E-G) Effect of hinge length on function of CD300a TASR. (E) Design of CD300a TASR variants with different hinge domains with the indicated amino acid lengths and their expression level after mRNA EP into B2M KO T cells. (F) NK challenge assay expressing the indicated CD300a TASR variant from panel E. N = 2 technical replicate curves per condition. (G) Correlation of NK protection from panel F as defined by IC50 value with hinge length from panel E. Error bar indicates 95% confidence interval of IC50 value. (H-J) Final optimization of CD300a TASR. (H) CD300a TASR V1 and 2 optimized variants are tested for expression via mRNA EP of B2M KO T cells. (I) NK challenge assay against the indicated CD300a TASR variant from (H). N = 1 technical replicate curve per condition. (J) Optimized CD300a TASR design and mechanism of action. GFP, green fluorescent protein; IgG, immunoglobulin G; ITIM, immunoreceptor tyrosine–based inhibitory motif.

Close Modal

or Create an Account

Close Modal
Close Modal