Bifunctional small molecules targeting PD-L1/CXCL12 as dual immunotherapy for cancer treatment

Inhibiting PD-1/PD-L1 interaction is a highly promising therapeutic modality. 1However, due to the low overall response rate in patients, researchers have attempted to combine PD-L1 inhibitors with other antitumor agents for cancer therapy.Studies have shown that combination immunotherapy of PD-L1 antibodies with CXCL12 inhibitors exhibited synergistic and better antitumor efficacy than monotherapy, indicating the potential clinical utility of targeting both PD-L1 and CXCL12 as dual immunotherapy to treat cancer. 2,3However, there are several disadvantages for combination therapy, including unpredictable PK/PD and overlapping toxicities.A potential alternative to combination therapy would be to use a single molecule with dual or multi-targeting capability, as the PK/PD of a single molecule is easily predictable.For example, dual-targeting bispecific antibodies (bsAbs) have gained significant attention in the field of anticancer drug discovery in recent years.Many PD-1/ PD-L1-based bsAbs (e.g., anti-PD-L1/TGF-β, anti-PD-1/CTLA-4, and anti-PD-1/LAG-3) have entered clinical trials as dual immunotherapy for treating cancer.However, bsAbs-based dual immunotherapies also suffer from the common drawbacks (e.g., immunogenicity, poor pharmacokinetics) as antibodies, thus it would be of high significance to develop small molecule PD-L1 inhibitor-based dual immunotherapy, as small molecules may overcome the above drawbacks of antibodies.We have previously reported PD-L1-targeting bifunctional molecules as potential anticancer agents. 4To continue our interest in this area, we designed a set of compounds targeting both PD-L1 and CXCL12 simultaneously as potential dual immunotherapy based on the hypothesis that PD-L1 and CXCL12 are two critical biomacromolecules controlling the immunosuppressive tumor microenvironment.Firstly, we analyzed the pharmacophores of PD-L1 inhibitors and CXCL12 inhibitors (Fig. 1a).The tail group of PD-L1 inhibitors and the hydroxyl moiety of CXCL12 inhibitors were exposed to solvent, making them suitable sites for conjugating the two inhibitors via a linker.Thus, twentyone bifunctional molecules were designed, synthesized (Supplementary Scheme S1), and bioevaluated (Supplementary Table S1).Among them, CP21 showed the strongest PD-L1-inhibitory effects with IC 50 of 78.6 nM (HTRF assay).Furthermore, CP21 displayed similar binding affinity (SPR assay) to both h(human)PD-L1 (K D = 66.9 nM, Fig. 1b) and m(mouse)PD-L1 (K D = 70.1 nM) (supplementary Fig. S1a).In addition, CD (Circular dichroism) assay revealed that when hPD-L1 or mPD-L1 was mixed with CP21, the conformation of their secondary structures changed similarly, as compared to the vehicle which contains only hPD-L1 (Fig. 1c) or mPD-L1 (Supplementary Fig. S1b).Moreover, the microscale thermophoresis (MST) assay confirmed that CP21 could bind to mPD-L1 with a K D of 654.1 nM (Supplementary Fig. S1c,d).Next, the binding affinity of CP21 to hCXCL12 and mCXCL12 was also determined by SPR and CD.CP21 bound to hCXCL12