Tumour inhibitory activity on pancreatic cancer by bispecific nanobody targeting PD-L1 and CXCR4

Abstract

Background: Antibodies and derivative drugs targeting immune checkpoints have been approved for the treatment of several malignancies, but there are fewer responses in patients with pancreatic cancer. Here, we designed a nanobody molecule with bi-targeting on PD-L1 and CXCR4, as both targets are overexpressed in many cancer cells and play important roles in tumorigenesis. We characterized the biochemical and anti-tumour activities of the bispecific nanobodies in vitro and in vivo. Methods: A nanobody molecule was designed and constructed. The nanobody sequences targeting PD-L1 and CXCR4 were linked by the (G4S)3 flexible peptide to construct the anti-PD-L1/CXCR4 bispecific nanobody. The bispecific nanobody was expressed in E. coli cells and purified by affinity chromatography. The purified nanobody was biochemically characterized by mass spectrometry, Western blotting and flow cytometry to confirm the molecule and its association with both PD-L1 and CXCR4. The biological function of the nanobody and its anti-tumour effects were examined by an in vitro tumour cell-killing assay and in vivo tumour inhibition in mouse xenograft models. Results: A novel anti-PD-L1/CXCR4 bispecific nanobody was designed, constructed and characterized. The molecule specifically bound to two targets on the surface of human cancer cells and inhibited CXCL12-induced Jurkat cell migration. The bispecific nanobody increased the level of IFN-γ secreted by T-cell activation. The cytotoxicity of human peripheral blood mononuclear cells (hPBMCs) against pancreatic cancer cells was enhanced by the molecule in combination with IL-2. In a human pancreatic cancer xenograft model, the anti-PD-L1/CXCR4 nanobody markedly inhibited tumour growth and was superior to the combo-treatment by anti-PD-L1 nanobody and anti-CXCR4 nanobody or treatment with atezolizumab as a positive control. Immunofluorescence and immunohistochemical staining of xenograft tumours showed that the anti-tumour effects were associated with the inhibition of angiogenesis and the infiltration of immune cells. Conclusion: These results clearly revealed that the anti-PD-L1/CXCR4 bispecific nanobody exerted anti-tumour efficacy in vitro and inhibited tumour growth in vivo. This agent can be further developed as a therapeutic reagent to treat human pancreatic cancer by simultaneously blocking two critical targets.

Nanobodies in cell-mediated immunotherapy: On the road to fight cancer

“…Using a microfluidic platform mimicking the TME, it was demonstrated the chemokine-nanobody fusion could selectively target PD-L1 + tumor cells and recruit effector cells to the TME ( 157 ). A unique anti-PD-L1/CXCR4 bispecific nanobody could successfully penetrate the tumor tissues in a xenograft mouse model and inhibit the growth of pancreatic cancer cells much better than the combination therapy using anti-PD-L1 and anti-CXCR4 nanobodies ( 158 ).…”

Small Antibodies with Big Applications: Nanobody-Based Cancer Diagnostics and Therapeutics

“…Several nanobodies against immune checkpoints have been developed, usually targeting single sites [107]. Recently, a nanobody molecule with bi-targeting on PD-L1 and CXCR4 were investigated in a pancreatic cancer since both of the targets are overexpressed in different cancers and play important roles in tumorigenesis [108]. The anti-PD-L1/CXCR4 bispecific nanobody consisted of sequences that target PD-L1 and CXCR4, linked by the (G4S) ×3 flexible peptide.…”

Advancements in Bispecific Antibody Development and Research Trends

“…scFv-HSA-scFv는 2개의 scFv 중간에 인간혈청알부민(human serum albumin)이 있는 구 조를 가지며, 알부민이 항체의 혈액내 반감기 및 안정성 증 가를 시킨다 [16]. Nanobody는 표적 항원에 결합하는 중쇄의 가변영역만 가진다 [17]. DART (Dual-affinity retargeting molecule)의 경우 기본적인 항체 구조와 유사하게 중쇄와 경 쇄로 구성되어 있으며 T 세포 활성화 결합부위가 존재하고, ImmTAC (Immune mobilizing monoclonal TCRs Against Cancer)은 T 세포 수용체(TCR)을 포함하여 이중특이성 항 체의 기능을 소화함과 동시에 T 세포 활성화도 유도하는 특 징을 가진다 [18,19].…”