Table 1.

Mouse TfR binding nanobody histidine mutants tested in vitro and in vivo

Nanobody	mTfR affinity pH 7.4 (nM)	Apparent mTfR unbinding ratio at pH 5.5 vs pH 7.4	NT-mediated Hypothermia (°C)
M1	5.11	1.01	0
M1: P96H	7.031	1.46	1
M1: Y98H	2509	1.28	2.5
M1: S99H	> 5000	1.01	1
M1: T100aH	55.31	1.05	1.5
M1: T100bH	30.12	1.24	0
M1: R100dH	1294	1.01	0
M1: L100eH	203	1.32	0
M1: D100fH	> 5000	1.58	0
M1: S100gH	> 5000	1.03	0
M1: K100iH	> 5000	1.42	0
M1: D101H	25	1.32	1.5
M1: Y32H, P96H	373	2.57	0
M1: G65H, P96H	> 5000	2.09	1
M1: P96H, Y98H	33.18	2.48	1.2
M1: P96H, S100H	1140	1.02	0
M1: P96H, D101H	242	2.65	7.5
M1: P96H, Y102H	11.65	2.33	6
M1: Y98H, Y102H	> 5000	1.29	5
M1: D31H P96H Y102H	95	1.13	0
M1: R33H P96H Y102H	1545	1.10	0
M1: R56H, P96H, Y98H	> 5000	0.98	1.5
M1: R56H, P96H, D101H	> 5000	1.78	0
M1: R56H, P96H, Y102H	6.653	2.63	8
M1: R56H, Y98H, D101H	52.6	1.04	0
M1: R56H, Y98H, Y102H	> 5000	0.99	0
M1: R56A, P96H, Y102H	1.7	2.59	0
M1: Y59H, P96H, Y102H	888	2.07	1
M1: A60H, P96H, Y102H	> 5000	ND	0
M1: P61H, P96H, Y102H	148	2.13	1
M1: S62H, P96H, Y102H	> 5000	2.18	1.5
M1: A64H, P96H, Y102H	> 5000	1.79	0
M1: S70H, P96H, Y102H	78	1.64	0
M1: P96H S99H D101H	301	2.43	2
M1: P96H S99H Y102H	> 5000	2.52	2.5
M1: P96H, D101H, Y102H	> 5000	2.42	0
M1: C22A C92A P96H D101H	35.76	1.03	0
M1: C22A C92A P96H Y102H	17.56	1.07	0
M1: R56H, P96H, Y98H, Y102H	> 5000	1.00	0
M1: R56H, P96H, S99H, Y102H	246	2.04	5.5
M1: Y32H, R56H, P96H, S99H, Y102H	> 5000	1.11	0

In vitro assessments included a) ELISA-based affinity measurements vs. recombinant mTfR extracellular domain conducted at pH 7.4 at room temperature, and b) plate-based unbinding assays conducted by binding nanobodies to recombinant mTfR extracellular domain at pH 7.4, then washing at either pH 5.5 or pH 7.4 at room temperature. In vivo assessments consisted of blinded measurements of mouse peak hypothermia following iv injection of 25 nmol/kg of each of the nanobodies fused with neurotensin (NT) (n = 3–6 mice per construct). ND: not determined due to low binding