Blockade of dual immune checkpoint inhibitory signals with a CD47/PD-L1 bispecific antibody for cancer treatment

Abstract

Background: Even though PD-1/PD-L1 is an identified key "don't find me" signal to active adaptive immune system for cancer treatment, the overall response rate (ORR) for all cancer patients is still limited. Other effective therapeutic modalities to bridge the innate and adaptive immunity to improve ORR are urgently needed. Recently, CD47/SIRPα interaction is confirmed as a critical "don't eat me" signal to active innate immunity. However, the red blood cell (RBC) toxicity is the big concern for the development of CD47-based anti-cancer therapeutics. Methods: Here, we report the development of a CD47/PD-L1 bispecific antibody 6MW3211 to block both PD-1/PD-L1 and CD47/SIRPα signals, and studied the effects of 6MW3211 on anti-tumor immune functions in vitro and in vivo. The pharmacokinetic and toxicity profiles of 6MW3211 were evaluated in GLP non-human primate (NHP) studies. Results: The dual immune checkpoint inhibitory signaling blocker 6MW3211 shows high binding affinity to PD-L1 and low binding affinity to CD47. This inequivalent binding affinity design makes 6MW3211 preferentially bound to PD-L1 on tumor cells followed by disrupting the interaction of CD47/SIRPα. Complex structure determination and flow cytometry assay demonstrated that 6MW3211 has no binding to either human or rhesus monkey RBCs. 6MW3211 effectively blocked both PD-1/DP-L1 and CD47/SIRPα signaling and promoted macrophage phagocytosis of tumor cells. Potent therapeutic efficacies of 6MW3211 in three different mouse models were further observed. Moreover, 6MW3211 was demonstrated to have a fairly good safety profile in a GLP NHP study. In addition, multiplex fluorescent immunohistochemistry (mIHC) staining shows that PD-L1 and CD47 co-express on several different types of human tumor tissues. Conclusions: These results support the development of 6MW3211 for the treatment of PD-L1 and CD47 double positive cancers.

Keywords: Bispecific antibody; Immunity.; Immunotherapy; Phagocytosis; Therapeutic efficacy.

Figure 1

Generation and characterization of 6MW3211. (A) Schematic diagram of the CD47/PD-L1 bispecific antibody 6MW3211. (B) Binding affinity measurement of 6MW3211 to hPD-L1 was performed by BIAcore T200 system. The KD of 6MW3211 to hPD-L1 was 0.55nM. (C) Binding affinity measurement of 6MW3211 to hCD47 was performed by BIAcore S200 system. The KD of 6MW3211 to hCD47 was 4.16 μM. (D) Simultaneously binding of 6MW3211 to hPD-L1 and hCD47 measured by Octet RED96 system. Binding of 6MW3211 to human (E) and rhesus monkey (F) RBCs. Red blood cells were adjusted to 5 × 10⁶ cells/mL, and added into a U-shaped-bottom cell culture plate at 100 µL/well. 5-fold serially diluted antibodies starting from 660 nM were added to cells. After that, cells were washed and added with PE-babbled goat anti-human IgG Fc and analyzed with flow cytometry. (G) Binding of 6MW3211 to hPD-L1 overexpressing HEK293-GFP (HEK293-hPD-L1-GFP) cells.

Figure 2

Decoding the interaction of 6MW3211-CD47 Fab with hCD47. (A) Overall structure of 6MW3211-CD47 Fab in complex with hCD47. (B) Interaction of CDR regions of Heavy and Light chains of 6MW3211-CD47 Fab with β-strand C, β-strand C', C'C“ loop and FG loop of CD47. (C) Hydrogen bonding interaction of Heavy chain CDR1 and CDR3 with CD47. (D) Hydrophobic interaction of Heavy chain CDR1 and CDR3 with CD47. (E) Interaction of Light chain CDR1 and CDR2 with CD47. Green and sky blue represent the Heavy and Light chains of 6MW3211-CD47 Fab, respectively, and gray represents CD47. The residues involved in the interaction are shown in a rod model, and the hydrogen bonding is indicated by blue dashed lines. (F) Superimposing CD47 of CD47/SIRPα complex (PDB code: 2JJS) with CD47 of 6MW3211-CD47 Fab/CD47 complex (PDB code: 7XJF). Grey represents CD47, pink represents SIRPα. (G) The detail interaction of 6MW3211-CD47 Fab with hCD47 nearby residue N32. (H) The detail interaction of 6MW3211-CD47 Fab with hCD47 nearby residue N55.

Figure 3

6MW3211 disrupting both CD47/SIRPα and PD-1/PD-L1 signals and promoting macrophage phagocytosis of tumor cells. (A) 6MW3211 binding to MDA-MB-231 tumor cells. 6MW3211 exhibited quite strong binding to MDA-MB-231 cells, with EC₅₀ of 0.53 nM, similar to the parental anti-PD-L1 antibody. (B) 6MW3211 blocking the interaction of PD-1 and PD-L1 performed in ELISA. (C) 6MW3211 disrupting the PD-1/PD-L1 signaling in reporter system. (D) 6MW3211 blocking the interaction of CD47/SIRPα performed in ELISA. (E) Macrophage phagocytosis of Raji and Raji-hPD-L1 cells mediated by 6MW3211. The concentrations for all antibodies used in this assay was 132 nM. Significance was calculated by two-way ANOVA with Sidak's multiple comparisons test. “*” means p < 0.05.

Figure 4

Anti-tumor activity of 6MW3211 in different mouse models. (A) The schematic diagram of the study design of Raji-luc/NCG model. 6MW3211 with different doses (0.04, 0.2, 1.0 and 5.0 mg/kg) or control antibody (hIgG4, 5.0 mg/kg) were administrated 3 days after Raji-luc cells injection. (B) Bioluminescence imaging of mice treated with 6MW3211 or control IgG. (C) Quantified bioluminescence signals for mice treated with 6MW3211 or control IgG. P value was labeled. (D) Kaplan-Meier survival curves of the Raji-luc cells in mice treated with the indicated doses of 6MW3211. (E) The schematic diagram of the study design of Raji-hPD-L1-luc/B-NDG model. 6MW3211 (1.0 or 5.0 mg/kg), the monovalent parental anti-CD47 arm (6MW3211-CD47-single) or control antibody (5.0 mg/kg) were administrated 3 days after Raji-hPD-L1-luc injection. (F) Bioluminescence imaging of mice treated with 6MW3211 or control IgG. (G) Quantified bioluminescence signals for mice treated with 6MW3211 or control IgG. P value was labeled. (H) Kaplan-Meier survival curves of the Raji-hPD-L1-luc cells in mice treated with the indicated doses of 6MW3211. (I) The schematic diagram of the study design of MC38-hPD-L1/hCD47 (B-hPD-L1/hCD47/hSIRPα) model. 6MW3211 with different doses (0.5, 2.0, 8.0 and 20.0 mg/kg) or control antibody (hIgG4, 20.0 mg/kg) were injected for 4 times. (J) Growth curves of MC38-hPD-L1/hCD47 tumors in B-hPD-L1/hCD47/hSIRPα mice treated with indicated doses of 6MW3211. (K) Photos of excised tumors treated with 6MW3211. (L) Tumor weight on day 33. The data reported as a Mean ± SEM and the significance was calculated by two-way ANOVA with Tukey's multiple comparison test compared with hIgG4. “*” means p < 0.05; “**” means p < 0.01 and “***” means p < 0.001.

Figure 5

Tissue distribution of 6MW3211 in MC38-hPD-L1/hCD47 tumor-bearing mice. (A) Coronal maximum intensity projection images of tumor-bearing mice treated with single intravenous administration of ⁸⁹Zr labeled 6MW3211 (200 μg/mouse) at different time points. (B) The tumor-to-muscle versus time curve in MC38-hPD-L1/hCD47 tumor-bearing mice after a single intravenous administration of ⁸⁹Zr labeled 6MW3211 (n = 6). The average tumor-to-muscle ratio showed a trend of rising at first, followed by a decline, reaching its peak value of 11.28 at 120 h post-dose. The tumor/muscle values were showed as Mean ± SME.

Figure 6

Pharmacokinetic and toxicokinetic profile of 6MW3211 in rhesus monkeys. (A) Mean PK profile of 6MW3211 in rhesus monkeys. A single dose of 6MW3211 (3, 10 and 30 mg/kg) were intravenously administered to rhesus monkeys, and the serum samples at different time points were collected and tested. (B) The schematic diagram of NHP toxicity study of 6MW3211. (C) The changes of RBCs in response to treatment with different doses of 6MW3211. (D) The changes of hemoglobin (HGB) in response to treatment with different doses of 6MW3211. (E) The changes of hematocrit (HCT) in response to treatment with different doses of 6MW3211. (F) The changes of reticulocyte (RET) in response to treatment with different doses of 6MW3211. All data values showed as Mean ± SEM.

Figure 7

Detection of the co-expression of CD47 and PD-L1 in different types of tumor tissues. (A-D) Multiplex immunofluorescence images displaying the co-expression PD-L1 and CD47 in ovarian cancer, lung cancer, bladder cancer and breast cancer tissues. The scale bars represent 200 μm and 10 μm, respectively. (E-H) Expression of PD-L1 and CD47 in different types of tumor tissues. Data points, each representing one tumor tissue, are shown by solid circles.