Structural characterization of the unique pH-responsive anti-TIGIT therapeutic antibody Ociperlimab

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28 Research Frontiers 2024 Research Frontiers 2024 Life Science TIGIT has attracted tremendous attention in cancer immunotherapy. As an inhibitory immune checkpoint, TIGIT is expressed on most NK and multiple T cell subsets and exerts inhibitory effects on innate and adaptive immunity by binding to its ligands, PVR (CD155) and nectin-2 [1]. Competitive binding of TIGIT and CD226 (an activating receptor) to PVR has been known as a key mechanism of TIGIT-driven immune suppression, and anti-TIGIT blocking mAbs are presumed to reverse the suppression by inhibiting TIGIT binding to PVR, thereby enhancing T cell or NK cell activity (Fig. 1 and Fig. 2(a)). Several clinical trials are currently evaluating the efficacy of anti-TIGIT mAbs in patients with different types of cancer. Ociperlimab from BeiGene and Tiragolumab from Roche block the TIGIT-PVR interaction and are in clinical development. However, the molecular blockade mechanism of these anti-TIGIT mAbs remains elusive. We determined the crystal structures of the Ociperlimab_Fab/TIGIT complex and the Tiragolumab_ Fab/TIGIT complex at resolutions of 2.4 and 2.0 Å with SPring-8 BL45XU beamline, respectively [2]. C o m p e t i t i v e b i n d i n g o f O c i p e r l i m a b _ F a b o r Tiragolumab_Fab against PVR was revealed by overlaying Ociperlimab_Fab/TIGIT or the Tiragolumab_ Fab/TIGIT complex on the PVR/TIGIT complex (Fig. 1). The binding of TIGIT by Ociperlimab_ Fab was dominated by the front CC’C’’ β -sheets, which have obvious steric clashes with PVR (Fig. 1 and Fig. 2(d)). The overlapping binding surface of Ociperlimab_Fab and PVR on TIGIT is located near the front CC’C’’ β -sheets. Similarly, the binding of TIGIT by Tiragolumab_Fab is also mediated by the front CC’C’’DF β -sheets. Moreover, the blockade mechanism of Ociperlimab_Fab relies on the steric hindrance of both the VH and VL domains against PVR to abolish its binding to TIGIT, whereas only the VH domain of Tiragolumab_Fab participated in the steric conflict with PVR to prevent its binding. Ociperlimab and Tiragolumab exhibit distinct binding modes toward TIGIT (Fig. 2(c)). We did not observe any obvious conformational change in TIGIT upon Ociperlimab_ Fab or Tiragolumab_Fab binding compared with its apo structure (Fig. 2(b)), as both antibodies mainly bind to the β -sheet of TIGIT with rigid secondary structures (Figs. 2(c,d)). However, Ociperlimab and Tiragolumab have distinct epitopes when binding to TIGIT, implying that the antagonistic mechanism of the two antibodies could be different. Almost all known pH-responsive antibodies sense pH due to histidine residues. pK a value of the histidine side chain is about 6. Thus, at pH 6.0 or below, the histidine side chain is mostly protonated whereas at physiological pH 7.4 it is deprotonated. As a result, the histidine side chain could change ionization states due to subtle changes in pH, leading to its net charge varying [3]. In some cases, histidine residues in the epitopes could also contribute to the pH-responsive antibodies as demonstrated by this structural study. Our surface plasmon resonance (SPR) data shows that the binding affinity of Ociperlimab towards TIGIT increased approximately 17-fold when the pH decreased from 7.4 to 6.0, whereas Tiragolumab did not show obvious pH- dependent binding to TIGIT (Fig. 3(c)). This observation is further supported by the fluorescence-activated cell sorting experiment which shows the enhanced binding of Ociperlimab to TIGIT when the pH shifts from 7.4 to 6.0 (Fig. 3(d)). In the Ociperlimab_Fab/TIGIT crystal structure, Asp103 HCDR3 is well positioned with respect to His76 TIGIT at a 2.7 Å distance and forms a strong electrostatic interaction, whereas no favorable architecture surrounding His76 TIGIT in Tiragolumab_ Fab/TIGIT was observed (Figs. 3(a,b)). Consistent with the critical role of His76 TIGIT in Ociperlimab binding, a substantial decrease in the binding affinity Structural characterization of the unique pH-responsive anti-TIGIT therapeutic antibody Ociperlimab Fig. 1. Competitive binding of Ociperlimab_Fab or Tiragolumab_Fab against PVR for TIGIT. (a) Superposition of Ociperlimab_Fab/TIGIT with the PVR/TIGIT (PDB: 3UDW) complex when aligned via TIGIT. (b) Superposition of Tiragolumab_Fab/TIGIT with the PVR/TIGIT complex when aligned via TIGIT. (c) The binding surface of TIGIT with PVR or Ociperlimab_Fab. (d) The binding surface of TIGIT with PVR or Tiragolumab_Fab. (a) (b) (c) (d) Ociperlimab Ociperlimab unique H chain H chain L chain L chain PVR PVR TIGIT TIGIT PVR unqiue Overlap Tiragolumab K82 K82 Tiragolumab unique Overlap PVR unqiue S80 S80 Q64 Q64 L65 L65 E60 E60 N58 N58 D63 D63 S78 S78 H76 H76 W75 W75 I68 I68 Q56 Q56 T55 T55 T112 T112 D115 D115 P114 P114 Y113 Y113 M23 M23 T117 T117 G116 G116 H111 H111 G74 G74 L73 L73 N70 N70 P79 P79 K82 S80 L65 L65 E60 E60 N58 N58 S78 H76 H76 I68 I68 I77 Q56 Q56 T55 T55 T112 T112 D115 D115 P114 P114 Y113 Y113 M23 M23 T117 T117 G116 G116 H111 H111 G74 G74 L73 L73 N70 N70 P79 29 Research Frontiers 2024 Research Frontiers 2024 of Ociperlimab to H76A TIGIT mutant was observed at pH 6.0, with a nearly 24,303-fold reduction (Fig. 3(c)). Meanwhile, Ociperlimab displayed comparable binding affinities to H76A TIGIT mutant at pH 6.0 and pH 7.4, confirming the critical role of His76 TIGIT in mediating this pH-dependent effect for Ociperlimab. These findings clearly demonstrate that His76 of TIGIT is indeed pivotal for the pH-dependent binding characteristics of Ociperlimab. Therefore, Ociperlimab has a stronger binding affinity with TIGIT under acidic pH conditions than physiological conditions, which could be valuable in targeting acidified tumor microenvironment and reducing on-target off-tumor toxicities. In summary, our structural studies on Ociperlimab and Tiragolumab have revealed the molecular blockade mechanism of these antibodies on TIGIT-driven immune suppression. The Ociperlimab competes with PVR towards TIGIT, which is consistent with previous functional studies by Chen et al . [4]. In addition, the pH-responsive property of Ociperlimab, rationalized in our structures, could potentiate the cytotoxicity of immune cells toward cancer cells in the tumor microenvironment. Therefore, the present complex structures should provide useful insights into improving the effectiveness of immunotherapeutic antibody development. References [1] X. Yu et al. : Nat. Immunol. 10 (2009) 48. [2] J. Sun, X. Zhang, L. Xue, L. Cheng, J. Zhang, X. Chen, Z. Shen, K. Li, L. Wang, C. Huang, J. Song: Structure 32 (2024) 550. [3] W. Wei et al. : Proteins 90 (2022) 1538. [4] X. Chen et al. : Front. Immunol. 13 (2022) 828319. Xiangxiang Zhang and Jian Sun* Dept. Biologics, BeiGene (Beijing) Co., Ltd., China *Email: jian1.sun@beigene.com Fig. 3. Structural basis of the pH- dependent property of Ociperlimab towards TIGIT. (a) The surrounding r e s i d u e s o f H I S 7 6 T I G I T w i t h i n Ociperlimab. (b) The surrounding r e s i d u e s o f H I S 7 6 T I G I T w i t h i n Tiragolumab. (c) SPR binding kinetics of the interactions of Ociperlimab or Tiragolumab with TIGIT at different pH values. ( d) Cell binding affinity measurement on Ociperlimab or Tiragolumab toward TIGIT under different pHs. Fig. 2. Mechanisms of action (MOA) of anti-TIGIT therapeutic antibodies. (a) A schematic diagram for MOA of anti-TIGIT antibodies. (b) Superposition of Fab-bound TIGIT with apo TIGIT (PDB: 3UCR). (c) Superposition of Ociperlimab_ Fab/TIGIT with the Tiragolumab_ Fab/TIGIT complex. (d) Structural overview of the TIGIT IgV domain. (a) (b) (c) (d) Ociperlimab Ociperlimab Ociperlimab unique Tiragolumab Tiragolumab unique Overlap NK/T NK/T CD226 TIGIT PVR CD226 TIGIT PVR Tumor cell Apo TIGIT Ociperlimab bound TIGIT (crystal form I) Ociperlimab bound TIGIT (crystal form II) Tiragolumab bound TIGIT N C C C F G N Y113 C’ B’ C’’ C’C’’ loop FG loop D B E A’ A TIGIT 90° 90° K82 P79 S78 I77 W75 G74 H76 L73 L73 L65 E60 N70 I68 H76 I77 S78 S80 Q64 D63 K82 P79 N58 Q56 H111 (a) (b) (c) (d) Ociperlimab TIGIT binding kinetics with Ociperlimab under different pHs TIGIT binding kinetics with Tiragolumab under different pHs Tiragolumab T31 W50 F104 Y106 D107 L108 L109 S32 Y91 D103 H76 H76 Ab Concentration ( μ μ g/ml) MFI 0 10 –4 10 –3 10 –2 10 –1 10 0 10 1 10 2 200 400 600 800 1000 1200 Ociperlimab pH 7.4 Ociperlimab pH 6.0 Tiragolumab pH 7.4 Tiragolumab pH 6.0 Analyte Analyte TIGIT TIGIT ( (W WT T) pH 7.4 ) pH 7.4 TIGIT TIGIT ( (W WT T) pH 6.0 ) pH 6.0 TIGIT (H76A) pH 7.4 TIGIT (H76A) pH 7.4 TIGIT (H76A) pH 6.0 TIGIT (H76A) pH 6.0 2.4 E+5 2.4 E+5 2.94 E+6 2.94 E+6 N.A. N.A. N.A. N.A. 1.27 E 1.27 E – – 4 4 9.28 E 9.28 E – – 5 5 N.A. N.A. N.A. N.A. 5.32 E 5.32 E – – 10 10 3.16 E 3.16 E – – 11 11 9.79 E 9.79 E – – 7 7 7.68 E 7.68 E – – 7 7 Kon Kon (M (M –1 –1 s s –1 –1 ) ) Koff Koff (s (s –1 –1 ) ) Ociperlimab Ociperlimab KD (M) KD (M) Analyte Analyte TIGIT TIGIT ( (W WT T) pH 7.4 ) pH 7.4 TIGIT TIGIT ( (W WT T) pH 6.0 ) pH 6.0 TIGIT (H76A) pH 7.4 TIGIT (H76A) pH 7.4 TIGIT (H76A) pH 6.0 TIGIT (H76A) pH 6.0 1.75 E+5 1.75 E+5 9.05 E+5 9.05 E+5 6.81 E+5 6.81 E+5 7.57 E+5 7.57 E+5 1.03 E 1.03 E – – 4 4 4.60 E 4.60 E – – 4 4 1.41 E 1.41 E – – 4 4 2.42 E 2.42 E – – 4 4 5.88 E 5.88 E – – 10 10 5.08 E 5.08 E – – 10 10 2.06 E 2.06 E – – 10 10 3.20 E 3.20 E – – 10 10 Kon Kon (M (M –1 –1 s s –1 –1 ) ) Koff Koff (s (s –1 –1 ) ) Tiragolumab Tiragolumab KD (M) KD (M) Sample code Sample code Top, MFI Top, MFI RV of top RV of top ( (% %) ) EC EC 50 50 ( ( μ μ g/ml) g/ml) RV of EC RV of EC 50 50 ( (% %) ) Hillslope Hillslope Hillslope Hillslope ( (% %) ) RV of RV of R R 2 2 972.3 972.3 100 100 0.03512 0.03512 100 100 1.16 1.16 100 100 0.9976 0.9976 990.3 990.3 102 102 0.02298 0.02298 153 153 1.177 1.177 101 101 0.996 0.996 919.6 919.6 95 95 102 102 0.09067 0.09067 100 100 1.196 1.196 103 103 0.9965 0.9965 988 988 0.1407 0.1407 64 64 1.047 1.047 90 90 0.9975 0.9975 Ociperlimab Ociperlimab pH 7.4 pH 7.4 Ociperlimab Ociperlimab pH 6.0 pH 6.0 Tiragolumab Tiragolumab pH 7.4 pH 7.4 Tiragolumab Tiragolumab pH 6.0 pH 6.0