PD‐L1/TIGIT bispecific antibody showed survival advantage in animal model

Dear Editor, Although PD-1 and PD-L1 (programmed cell death1/ligand 1) antibody therapies have provided persistent benefits in various cancers, their clinical applications have been limited by relatively low response rates and the occurrence of drug resistance.1 Thus, they have been extensively investigated in combination with a variety of antitumor therapies, such as inhibitors to other immune checkpoints in combinational or bispecific antibody (BsAb) therapeutical strategies, to improve the response rates or efficacies in the clinic.2 TIGIT (T-cell immunoreceptor with immunoglobulin and ITIM domains) is an inhibitory T-cell immunoreceptor discovered in 2009,3 which is expressed mainly on regulatory T cells (Tregs), memory T-cells, and NK cells. Combination therapies that block both PD-L1 and TIGIT pathways have superior clinical benefits to PD-L1 monotherapy.4 BsAbs simultaneously targeting both pathways are also entering clinical trials (NCT05102214), but whether BsAbs are superior to combinational approaches has not been elucidated yet. We reported here a novel bispecific anti-PD-L1/TIGIT antibodywith potent effects in enhancing human IL-2 production by primary human T cells in vitro and in increasing the overall survival in a transgenic mouse model in vivo.More importantly, we compared the tetravalent bispecific anti-PD-L1/TIGIT antibody to mAb and combination therapies head-to-head systematically, and our data supported the the advantage of taking bispecific approach over mAb and combination therapies experimentally. First, hybridomas were established5 and humanization was carried out through "framework shuffling6,7 on both anti-PD-L1 and anti-TIGIT antibodies. Both chimeric (antiPD-L1-8A1 and anti-TIGIT-31C3) and humanized (antiPD-L1-#77, anti-TIGIT-T26 and T87) antibodies showed binding and blocking capabilities comparable to corresponding positive controls (Figure 1A–D), i.e., atezolizumab (Tencentriq) for anti-PD-L1 and tiragolumab for


PD-L1/TIGIT bispecific antibody showed survival advantage in animal model
Dear Editor, Although PD-1 and PD-L1 (programmed cell death-1/ligand 1) antibody therapies have provided persistent benefits in various cancers, their clinical applications have been limited by relatively low response rates and the occurrence of drug resistance. 1 Thus, they have been extensively investigated in combination with a variety of antitumor therapies, such as inhibitors to other immune checkpoints in combinational or bispecific antibody (BsAb) therapeutical strategies, to improve the response rates or efficacies in the clinic. 2 TIGIT (T-cell immunoreceptor with immunoglobulin and ITIM domains) is an inhibitory T-cell immunoreceptor discovered in 2009, 3 which is expressed mainly on regulatory T cells (Tregs), memory T-cells, and NK cells. Combination therapies that block both PD-L1 and TIGIT pathways have superior clinical benefits to PD-L1 monotherapy. 4 BsAbs simultaneously targeting both pathways are also entering clinical trials (NCT05102214), but whether BsAbs are superior to combinational approaches has not been elucidated yet.
We reported here a novel bispecific anti-PD-L1/TIGIT antibody with potent effects in enhancing human IL-2 production by primary human T cells in vitro and in increasing the overall survival in a transgenic mouse model in vivo. More importantly, we compared the tetravalent bispecific anti-PD-L1/TIGIT antibody to mAb and combination therapies head-to-head systematically, and our data supported the the advantage of taking bispecific approach over mAb and combination therapies experimentally.
To obtain a well-behaved anti-PD-L1/TIGIT molecule, eight BsAb antibodies (Bs#1-Bs#8) containing anti-TIGIT-huT26 or huT87 in IgG4, and anti-PD-L1-hu#77 in scFv were generated ( Figure S5A). Additionally, the sequences of VH and VL in the scFv segment (VH-linker-VL and VL-linker-VH) with and without an additional disulphide bond mutation were taken into consideration. 8 We measured the binding kinetics of eight BsAbs by BLI technology on Octet 9 to both human PD-L1-his and human TIGIT-his, which indicated that the affinities to human PD-L1-his (6-9 nM) ( Table 1) slightly decreased compared to humanized mAb (2.66 nM), but affinities to TIGIT were not affected ( Figure S1).
The binding and neutralization assays of eight BsAbs were analyzed by ELISA (Figure 2A Anti-PD-L1-hu#77 was administered i.p. at 1 and 5 mg/kg, and atezolizumab analogue at 5 mg/kg every 3 days. Tumour volume was monitored using electronic callipers every other day. (H) MC38 tumour cells inoculated in C57BL/6-HU-TIGIT transgenic mice. Anti-TIGIT was injected i.p. at 5 mg/kg and tumour size was measured every 3 days. Tumour volumes were presented as mean ± SEM. Statistical significance (p-value) was determined using one-way or two-way ANOVA with Tukey's multiple comparison test (*p < .05, **p < .01, ***p < .001) (Tables S1 and S2). However, Bs#5 to Bs#8 with the extra disulphide bond mutation had higher binding abilities on FACS. Bs#5 had the highest binding activities, with EC 50 values of 0.85 nM for HEK293-hPD-L1 and 0.55 nM for HEK293-hTIGIT.
Next, the purity and thermostability of BsAbs were evaluated through the HPLC-SEC method with native (nontreated) and heat-treated (60 • C in a water bath for 1 h) BsAbs. The monomer ratios in native state and the decrease between the non-treated and heat-treated BsAbs revealed that BsAbs with a disulphide bond mutation in scFv (Bs#5 to Bs#8) had better purity (Table 1; Figure  S5B). The disulphide bond mutation favoured the VHlinker-VL orientation in our BsAbs by comparing Bs#5 (2.5% decrease) to Bs#1 (11.7%), and Bs#6 (10.0% decrease) to Bs#2 (3.8%). Among them, Bs#5 with additional disulphide bond and in VH-linker-VL sequence showed better behaviour in purity and thermostability than other BsAbs.  Based on our results, Bs#5 was selected as the BsAb candidate for further evaluation. The affinities and dual binding activity of Bs#5-hIgG1 (Bs#5.1) were detected by BLI ( Figure 3A-D), which exhibited high affinities to both targets and outstanding dual-binding ability regardless of loading sequences of two antigens. Then, the biological functions of Bs#5.1 were explored in the SEB-activated PBMC experiment in vitro and double-gene humanized mouse model (C57BL/6-HU-PD-L1/TIGIT) in vivo. In the PBMC activation assay, Bs#5.1 increased the human IL-2 secretion sitmualted by SEB to the highest level ( Figure 3E-H). In the animal study, Bs#5.1 treatment extended the survival time to the greatest extent and was the only group that showed a significant difference in overall survival compared to the isotype group ( Figure 3I,J). However, the BsAb treatment failed to achieve a significant difference in tumour volume due to the great variability within the group. The tumour growth curves were displayed in Figure S6. The variability might arise from the relatively low dose regimen and the heterogenicity of hPD-L1 expression on MC38-hPD-L1 cells ( Figure S3).

TA B L E 1 KD values and monomer ratios of bispecific antibodies (BsAbs)
Finally, pharmacokinetics (PK) analysis 10 of Bs#5.1 was conducted in four female transgenic mice following a single dose injection of 10 mg/kg i.v., which showed similar PK curves by two ELISA methods, indicating that Bs#5.1 was intact and stable in mouse serum and capable of binding both targets ( Figure S7).
Currently, several bispecific antibodies targeting PD-L1/TIGIT (such as HLX301 from Henlius, Inc. and PM1022 from Biotheus Inc.) or PD-1/TIGIT (IBI321, jointly developed by Eli Lilly and Innovent Biologics, Inc.) have been advanced to Phase I clinical trials with no clinical antitumor efficacies reported yet. The combination therapy with PD-L1 and TIGIT inhibitors has demonstrated clinical benefits (CITYSCAPE study from Roche) in Phase II clinical trials. However,whether bispecific antibodies targeting PD-1 (or PD-L1) and TIGIT have advantage over mAb and combination therapies has not been demonstrated.
Herein, we constructed a BsAb targeting both PD-L1 and TIGIT in the format of IgG-scFv and systematically characterized its functions in vitro and in vivo. BsAb induced the highest level of IL-2 production in primary human T cells when compared to the mAbs and combination. In the transgenic animal model, BsAb increased the overall survival to the greatest extent. Taken together, our data favour the BsAb approach over mAb and combination therapies.