Overcoming antigen tolerance to develop GB22-45-2, an anti-DKK1 antibody for gastric cancer
Wenjun Zhang1, Xiling Wei1, Tianqi Yao1
1Centre for Research and Development, Kexing Biopharm Co., Ltd, Shenzhen, Guangdong 518057, China.
Background:
Gastric cancer (GC) represents one of the most prevalent and lethal malignancies, however, current treatments have shown limited efficacy. Clinical investigations have revealed that Dickkopf-1 (DKK1)-high GC patients are associated with poor prognosis, and DKK1 expression is negatively correlated with overall survival, suggesting a means of pharmaceutical intervention by neutralizing DKK1 for GC patients.
Methods:
To overcome antigen tolerance and stimulate antibody production, strategies were employed, including different adjuvants, antigen design, and various mouse strains. Finally, a novel antibody targeting DKK1 was screened out from ~100 000 candidates. GB22-45-2 underwent in vitro and in vivo evaluations, and was compared with clinical-stage anti-DKK1 benchmarks. A comprehensive assessment of drug developability was also performed.
Results:
GB22-45 screened out from Murphy Roths Large (MRL/MpJ) mice immunized with Freund's adjuvant and full-length DKK1 conjugated with Keyhole Limpet Hemocyanin achieved the best affinity. C34V and M99V mutations were introduced to the final optimized humanized antibody, GB22-45-2, to prevent dimer formation and mitigate risks of methionine oxidation. In vitro, GB22-45-2 exhibited potent activities in restoring Wnt T-cell factor/lymphoid enhancer-binding factor signaling by reporter assay. Importantly, GB22-45-2 demonstrated a desirable specificity profile by baculovirus particle binding assay and cross-interaction chromatography assays. In vivo, GB22-45-2 showed a modest tumor growth inhibition (31.03%) that is significant (P = 0.0175), while DKN-01 did not reach significance, in the MKN-45-Balb/c-nude mouse model. Additionally, GB22-45-2 exhibited a satisfactory developability profile, including good thermal stability, low aggregation propensity, high structural integrity, and excellent stability under various stress conditions.
Conclusions:
In summary, these preclinical results suggest that GB22-45-2 is a potential therapeutic candidate for GC, laying the foundation for its future drug development.
