Table 1.

Binding properties of RD1 library-derived TCR clones¹

TCR Variant	pepMHC	KD equilibrium (kinetic)	EC50 HLA-A2 monomer	EC50 HLA-A2 Ig dimer
RD1-Tax-1	Tax/HLA-A2	84 nM (34 nM)	297 nM	110 nM
RD1-MART1	MART1/HLA-A2	3.1 ± 3.9 μM	> 5.0 μM	130 nM
RD1-MART1HIGH	MART1/HLA-A2	250 ± 120 nM (68 nM)	52 nM	2 nM
T1-S18.45	MART1/HLA-A2	45 nM	167 nM	12 nM

¹SPR experiments were performed at 25°C with peptide/HLA-A2 monomers immobilized on a sensor chip and soluble scTCR flowed over in solution. For one replicate with RD1-MART1 and RD1-MART1^HIGH scTCRs, the orientation was reversed such that soluble scTCR was immobilized on a sensor chip and soluble pepMHC was flowed over in solution (curves shown in Supplementary Fig. 11). MART1 peptide used for these experiments utilized the anchor modified decamer MART1 variant (i.e. MART1_{26–35 A27L}: ELAGIGILTV). For equilibrium SPR values, n=4 for RD1-MART1 and n=3 for RD1-MA11RT1^HIGH. For kinetic SPR values, n=1 for A6-X15 and RD1-MART1^HIGH. EC₅₀ values for titrations on the surface of yeast with soluble peptide/HLA-A2 monomers and HLA-A2-Ig dimers are reported with n=1. A6-X15 TCR (sequence identical to RD1-Tax1) value previously measured to be 53.4 nM by SPR³⁴. T1-S18.45 is a MART1-specific TCR, previously engineered for higher affinity with a measured affinity of 45 nM²⁴. Typically EC50 values are higher than SPR valued at affinities >100 nM due to wash steps prior to flow cytometry analysis; A possible explanation for the discrepancy of RD1-MART1^HIGH is that the scTCR protein was less stable in solution at high concentrations.