Table 1.

Midpoint unfolding temperature (Tm) and binding affinity at 20 °C of V_HH-7D12 variants.

Variants	MW (Da)	Tm (°C)	SPR
			kon (M−1s−1)	koff (s−1)	KD(M)	KD(M) heat
VHH-7D12	13,635	63	3.2 × 105 ± 5.0 × 104	0.003 ± 9.7 × 10−5	1.1 × 10−8 ± 1.7 × 10−9	1.0 × 10−8 ± 1.8 × 10−10
7D12-C5R	14,588	61	4.1 × 105 ± 5.9 × 104	0.004 ± 3.8 × 10−5	9.7 × 10−9 ± 1.3 × 10−9	1.1 × 10−8 ± 5.9 × 10−10
7D12-C9R	15,269	61	4.5 × 105 ± 6.6 × 104	0.004 ±1.0 × 10−4	9.4 × 10−9 ± 1.0 × 10−9	1.0 × 10−8 ± 6.9 × 10−10
7D12-C5K	14,447	59	3.8 × 105 ± 3.4 × 104	0.003 ± 4.1 × 10−5	1.0 × 10−8 ± 1.0 × 10−9	1.1 × 10−8 ± 5.7 × 10−11
7D12-C9K	15,017	59	3.6 × 105 ± 1.4 × 104	0.004 ± 0.0001	1.2 × 10−8 ± 8.6 × 10−10	1.1 × 10−8 ± 7.3 × 10−11

^heat: SPR measured at 20 °C after incubation at 75 °C in PBS for 45 min. The SPR data are shown in Figure S5.