We have used P301S human tau transgenic mice (Yoshiyama et al., 2007) to test intracerebroventricular (ICV) administration of three different C646 supplier anti-tau antibodies selected for their ability to block tau seeding activity in vitro and to block tau uptake into cells. We have previously

observed that tau aggregates, but not monomer, are up taken by cultured cells and that internalized tau aggregates trigger intracellular tau aggregation in recipient cells (Frost et al., 2009 and Kfoury et al., 2012). We characterized the HJ8 series of eight mouse monoclonal antibodies (raised against full-length human tau) and HJ9 series of five antibodies (raised against full-length mouse tau) in an adapted cellular biosensor system we have previously described (Kfoury et al., 2012) that measures cellular tau aggregation induced by the addition of brain lysates containing tau aggregates. The click here antibodies had variable effects in blocking seeding, despite the fact that all antibodies efficiently bind tau monomer and stain neurofibrillary tangles. We selected three antibodies with different potencies in blocking seeding for our studies. Prior to testing in vivo, we

determined the binding affinities and epitopes of the antibodies, which are all IgG2b isotype. We immobilized human and mouse tau on a sensor chip CM5 for surface plasmon resonance (SPR) (Figure 1). The HJ9.3 antibody, raised against mouse tau, recognizes both human (Figure 1A) and mouse (Figure 1B) tau with the same binding constant (KD = Kd/Ka = 100 pM) (Figure 1G). The association (Ka) and dissociation (Kd) rate constants were calculated by using BIAevaluation software (Biacore AB) selecting Fit kinetics simultaneous Ka/Kd (Global fitting) with 1:1 (Langmuir) interaction model. The Ka and Kd of HJ9.3 toward human (Ka = 7.5 × 104 Ms−1, Kd = 7.5 × 10−6 s−1) and mouse (Ka = 8.6 × 104 Ms−1, Kd = Thalidomide 9.1 × 10−6 s−1) indicate strong binding to both. We mapped the epitope of HJ9.3 to the repeat domain (RD) region, between amino acids 306–320. HJ9.4, raised against mouse tau, had high affinity KD (2.2

pM) toward mouse tau with a high association rate constant (Ka = 2.28 × 105 Ms−1) and very low dissociation constant (Kd = 5.1 × 10−7 s−1) ( Figures 1D and 1G). However, the same antibody had a much lower affinity (KD = 6.9 nM) toward human tau ( Figures 1C and 1G), with a similar association rate constant (Ka = 1.5 × 105 Ms−1) as mouse tau but with much faster dissociation (Kd = 1.07 × 10−3 s−1). Thus, the HJ9.4 interaction with human tau is less stable than with mouse tau. The epitope for this antibody is amino acids 7–13. HJ8.5 was raised against human tau. It binds to human tau ( Figure 1E) but not to mouse tau ( Figure 1F). The KD (0.3 pM) ( Figures 1E and 1G) and low dissociation rate (Kd = 4.38 × 10−8 s−1) indicate that HJ8.5 binds human tau with very high affinity. We mapped the epitope of HJ8.