Crystallisation of the anti-pTau C21-ABS Fab in complex with pTau reveals an unchanged paratope
To investigate how ultra-humanisation of anti-pTau altered the paratope and epitope interface, we generated purified anti-pTau C21-ABS Fab (Fig. S1 and S2A) and crystallized it in complex with the pTau peptide (Fig. S2B). Prior to Fab generation, we confirmed, by SPR analysis with immobilized pTAU peptide, that the binding affinity of both WT and C21 anti-pTau mAbs were comparable (data not shown), as has been previously reported 19.
The sequence of the pTau peptide (224KKVAVVR(pT231)PPK(pS235)PSSAKC241) was the same as that previously crystallized in complex with anti-pTau WT Fab (Protein Data Bank ID 4GLR) 20. The structure of the anti-pTau C21-ABS Fab in complex with the pTau peptide was solved to a resolution of 2.6 Å (Table S1). Two complexes are present in the asymmetric unit, displaying identical binding modes. Analysis is presented of chains H, L and P.
In the anti-pTau C21-ABS Fab-pTau co-crystal structure, 10 amino acids from the pTau peptide are visible (224KKVAVVR(pT231)PP233) (Fig. 1A and B). The remaining 8 (234K(pS235)PSSAKC241) are disordered. The positioning of the pTau peptide is identical in the anti-pTau WT Fab and ABS-C21 co-crystal structures (Fig. S3). The pTau peptide is positioned above the heavy chain CDRs (Fig. 1A and 1B) with two sharp turns at Val228pTau and pThr231pTau (Fig. S4).
Comparative analysis of the two crystal structures (WT and ABS-C21) shows that the pTau epitope remains unchanged, comprising 225KVAVVR(pT) 231 (Fig. 1C and D). In both structures, the Fab fragment recognizes six amino acids N-terminal to the Thr231 phosphorylation site via CDR-H3, with CDR-H2 dominating the phospho-epitope through interaction with pThr231pTau itself. CDR-H1 and the light chain CDRs provide secondary support (Fig. 1C and 1D). The anti-pTau paratope structure remains similar after the ultra-humanisation process with very few individual interactions disrupted or altered. Overall, there is a removal of 1 x VH (S52aCDRH2) and 2 x VL (Y31CDRL1 and W50CDRL2) interactions in the paratope after ultra-humanisation of anti-pTau WT to anti-pTau C21-ABS (Fig. 1C and D). Additionally, there is a change in the type of molecular interaction between LCDR1 and HCDR2 as a result of the amino acid substitutions arising from germlining. The mutation D32YCDRL1 results in a change from a salt-bridge in wild-type to hydrophobic interaction with K225pTau in C21.
In LCDR1, 4 out of 6 amino acid residues have been humanized, with the germline residue being positively selected (Table S2). Two of these, Y31WT and D32WT, were identified as contact points in the WT co-complex structure, forming a hydrophobic interaction with V228pTau and salt bridge interaction with K225pTau, respectively (Fig. 1C). In the ABS-C21 variant, these have been mutated to S31C21 and Y32C21 (Fig. 1D). This suggests that they are support residues rather than key contact points in the anti-pTau paratope. V228pTau interacts with two other anti-pTau residues (E100fCDRH3 and Y91CDRL3), thereby facilitating the Y31SCDRL1 mutation. Interestingly, the D32YCDRL1 mutation introduces a new hydrophobic interaction with K225pTau. In this way, the positioning of the pTau peptide is maintained. The loss of negative charge that occurs through the D32YLCDR1 mutation is not significant as the overall negative charge of this surface area is maintained by E100fHCDR3, which in turn maintains a salt-bridge interaction with K225pTau (Fig. S5).
2 out of 4 amino acid residues have been humanized in LCDR2 (Table S2). One of these, W50WT, was identified as a contact point in the WT paratope, forming a hydrophobic interaction with K225pTau. The W50G mutation disrupts this interaction by replacement of the bulky Trp side-chain with the much smaller Gly side-chain. However, K225pTau is positionally maintained through three interactions with HCDR3 (L97, Q100a and E100f) and one with LCDR1 (Y32), thereby facilitating the mutation of W50G without loss of antigen binding.
2 out of 9 chicken residues are mutated to germline amino acids in LCDR3. Importantly, Y91WT is not mutated, forming a hydrophobic interaction with V228pTau which remains undisrupted in the C21-ABS Fab-peptide co-complex structure. Interestingly, Y91 was the only LCDR residue that was 100% maintained in our previous analysis of selection output clones from the ultra-humanisation process 19. The preservation of this residue would suggest its intimate involvement in ligand binding, and this is confirmed with our structural analysis.
Overall, HCDR1 plays a support role in the anti-pTau paratope. However, neither of the two chicken residues present in this CDR are mutated to germline through the ultra-humanisation process (Table S2). The chicken residue Q33 is a key contact residue forming a side-chain hydrogen bond with V229pTau which is preserved in the anti-pTau C21-ABS structure. In agreement with this, we previously observed Q33 to be a “strongly maintained” residue after library selection, with 100% of output clones favouring this residue 19.
In CDRH2, 6 out of 11 amino acid residues have been humanized (Table S2). Of these, only S52a comprises a contact residue in the WT structure, forming a hydrogen bond with pThr231 (Fig. 1C). This has been mutated to Gly in the ABS-C21 structure, with the hydrogen bond being disrupted as a result of the Ser side chain being replaced with the much smaller Gly amino acid side chain. This has no effect on antigen affinity, however, as 3 other hydrogen bond interactions in total are formed between CDRH2 and pThr231 through T52C21, R53C21 and G55C21 (Fig. 1D). Interestingly, the S(52a)G mutation is not favoured in our original sequence analysis of ABS selection output clones, showing > 90% retention across the library. Although unfavoured, the bonds supplied by the neighbouring amino acid residues T52C21, R53C21 and G55C21 allow for this mutation (Fig. 1D).
No residues in HCDR3 are mutated and the overall loop structure remains identical to the WT structure. E100fWT maintains a critical salt bridge interaction with K225pTau and hydrogen bond with V228pTau, positioning the pTau peptide with a sharp turn at V228pTau (Fig. S3). Taken together, these observations confirm our previous hypothesis that only functionally required CDR content is retained after mAb ultra-humanisation.
Comparison of the anti-pTau WT and C21-ABS mAb surface charge distribution reveals the importance of electrostatic interactions in the phospho-epitope
To determine the percentage reduction in the presence of non-germline amino acid solvent accessible surface area (ngSASA) on the Fab fragment, jsPISA (Protein Interfaces, Surfaces & Assemblies) analysis was carried out using the CCP4 software suite 21. In comparison to the parental molecule, the ngSASA was reduced from 2599.9 Å2 (WT) to 1882.5 Å2 (C21-ABS). This is a difference of 27.5%, representing a significant reduction in the presence of non-germline amino acids on the surface of the anti-pTau C21 Fab fragment (Fig. 2A). Despite the significant reduction in ngSASA, key amino acid residues in the paratope remain unchanged. Very few molecular interactions are altered in the paratope following the ultra-humanization process (Fig. 1C and D). Importantly, the overall electrostatic surface-charge distribution of anti-pTau WT and C21-ABS also remains similar despite the reduction in ngSASA (Fig. 2B). This clearly demonstrates the importance of electrostatic interactions in pTau recognition by the anti-pTau Fab fragment. In total, 4 amino acids (R53CDRH2, D100CDRH3, E100fCDRH3 and D32LCDR1) in the anti-pTau WT paratope contribute to electrostatic interactions (Fig. 1C).
As observed in the anti-pTau WT structure, the pTau phosphorylation site pThr231 is exclusively recognized by CDR-H2, facilitated by the formation of a positively-charged pocket to accommodate the phosphate group 20. The positive charge in this pocket is contributed by R53C21 (Fig. S6). In our analysis of ABS selection output clones, R53C21 was 100% maintained across all clones, indicating that this residue is crucial to antigen specificity. This is clearly explained through the observation in both WT and ABS-C21 crystal structures that this residue makes up the positive charge in the CDRH2 pocket.
In CDRH3, D100C21 and E100fC21 form negatively charged patches positioned by the N-terminus of the pTau peptide (Fig. S6). D100C21 and E100fC21 form salt-bridge interactions with R230pTau and K225pTau, respectively (Fig. 1D). These residues are both maintained throughout the ultra-humanisation process, illustrating their importance in maintaining antigen affinity.
D32Y is the only amino acid mutation introduced by the ultra-humanisation process that disrupts an electrostatic interaction. This mutation disrupts a salt-bridge interaction with K225pTau. This is in agreement with our previous analysis of the ABS selection output clones in which D32WT had a ~ 50% retention rate, indicating that this residue is not crucial for antigen binding. In the anti-pTau ABS-C21 variant, this residue has not been retained. The K225pTau interaction with E100fC21 appears sufficient to maintain the positioning of the pTau peptide through a salt-bridge interaction with K225pTau (Fig. 1D). Furthermore, this residue maintains the negatively charged surface of the anti-pTau Fab required to accommodate K225pTau (Fig. S6).
The presence of in-silico predicted T-cell epitopes are reduced in anti-pTau C21by ABS
When analysing the immunogenic potential of a mAb, one of the first steps is to use in-silico tools to predict the presence of possible T-cell epitopes. For this application, software algorithms and machine-learning methods have been employed to carry out systematic assessments of MHC class II peptide-binding domains within proteins 22.
In-silico predictions for T-cell epitopes within the V-genes of anti-pTau WT and C21-ABS were previously performed using EpiMatrix (Epivax software, RI) 19 (Fig. 3A). Following ultra-humanisation of anti-pTau, the total number of predicted T-cell epitopes was reduced from 5 in anti-pTau WT (1 VL and 4 VH epitopes) to 1 (0 VL and 1 VH epitope) in anti-pTau C21-ABS. In our analysis, predicted T-cell epitopes are defined as peptides with four or more “hits” in the EpiMatrix assessment prediction of binding to each of 8 HLA Class II alleles assessed (DRB1*0101, DRB1*0301, DRB1*01, DRB1*0701, DRB1*0801, DRB1*1101, DRB1*1301, and DRB1*1501). 9-mer peptides are classified as “hits” when scoring an EpiMatrix assessment above 1.64 (approximately the top 5% of any given peptide set).
The remaining predicted T-cell epitope is present in HCDR1 (Fig. 3A). In HCDR1, Q33 and M35 are the only two chicken residues that were toggled in the ABS library for selection (Table S2). Both of these residues were > 90% maintained in our analysis of ABS selection output clones 19, indicating that they are important residues for antigen binding. The structure of anti-pTau C21-ABS in complex with the pTau peptide confirms that Q33 is a key contact residue, forming a side-chain hydrogen bond with V229pTau. Due to the importance of this interaction, it was therefore not possible to mutate this amino acid without disruption of antigen binding (Fig. 1C & D).
Predicted T-cell epitopes were mapped onto the anti-pTau C21-ABS and anti-pTau WT crystal structures, demonstrating a significantly reduced surface presence of amino acids in predicted T-cell epitope regions (Fig. 3B). Together, these in silico predictions indicate a lowered risk of immunogenicity in the ultra-humanised molecule anti-pTau C21-ABS. It is therefore likely that this ultra-humanised mAb will elicit a weaker immunogenic response in vivo, thus avoiding complications of ADA and reduced half-life.