Preclinical development of a novel CD47 nanobody with less toxicity and enhanced anti-cancer therapeutic potential

Abstract

Background: CD47, the integrin-related protein, plays an important role in immune resistance and escape of tumor cells. Antibodies blocking the CD47/SIRPα signal pathway can effectively stimulate macrophage-mediated phagocytosis of tumor cells, which becomes a promising approach for tumor immunotherapy. Nanobodies (Nbs) derived from camelid animals are emerging as a new force in antibody therapy.

Results: HuNb1-IgG4, an innovative anti-CD47 nanobody, was developed with high affinity and specificity. It effectively enhanced macrophage-mediated phagocytosis of tumor cells in vitro and showed potent anti-ovarian and anti-lymphoma activity in vivo. Importantly, HuNb1-IgG4 did not induce the agglutination of human red blood cells (RBCs) in vitro and exhibited high safety for hematopoietic system in cynomolgus monkey. In addition, HuNb1-IgG4 could be produced on a large scale in CHO-S cells with high activity and good stability. Also, we established anti-CD47/CD20 bispecific antibody (BsAb) consisted of HuNb1 and Rituximab, showing more preference binding to tumor cells and more potent anti-lymphoma activity compared to HuNb1-IgG4.

Conclusions: Both of HuNb1-IgG4 and anti-CD47/CD20 BsAb are potent antagonists of CD47/SIRPα pathway and promising candidates for clinical trials.

Keywords: Bispecific antibody; CD47; Immunotherapy; Nanobody.

Fig. 1

Anti-CD47 nanobody discovery. a SDS-PAGE of purified CD-47 ectodomain (ECD) antigen. Lane 1 was loaded with protein ladder; Lane 2 showed purified human CD47 (ECD)-Fc antigen. The samples were separated by using 10% gradient PAGE gels and stained with Coomassie blue. The experiment was performed in triplicate and one representative experiment was shown. b The enrichment fold for phage particles in wells coated with antigen versus wells without antigen was detected after each round of panning. The experiment was performed once. c The blocked nanobody was selected by FACS. The Nb1-IgG4 was identified that could block the interaction between human CD47 and SIRPα. The experiment was performed in triplicate and one representative experiment was shown

Fig. 2

Characterization of humanized Nb1-IgG4 (HuNb1-IgG4). a CDRs sequences of HuNb1-IgG4 were shown. b The activity of HuNb1-IgG4 binding to hCD47 on the surface of cells was determined by FACS. c The activity of HuNb1-IgG4 blocking CD47/SIRPα was determined by FACS. d The specificity of HuNb1 to human CD47, mouse CD47 and rat CD47 antigen was detected by ELISA. The above experiments were performed in triplicate and data were presented as mean ± SD. ***P < 0.001 versus control. e The affinity of B6H12 and HuNb1-IgG4 to CD47 was detected by Fortebio detection. The experiment was performed in triplicate and one representative experiment was shown

Fig. 3

HuNb1-IgG4 induces phagocytosis and shows high activity of anti-tumor in human ovarian tumor-engrafted mice models. a Representative result for phagocytosis of CFSE-labeled Jurkat E6.1 cells phagocytosed by macrophages. The results were analyzed by FACS and showed in a bar graph. The experiment was performed in triplicate and data were presented as mean ± SD. b BALB/c nude mice were subcutaneously transplanted with SKOV3 cells and treated with low (0.2 mg/kg), medium (1 mg/kg) and high (5 mg/kg) dose of HuNb1-IgG4 and high (5 mg/kg) dose of Hu5F9-G4 or PBS as the control (n = 8). Tumor tissues from all mice in each group were shown. c Tumor volumes were measured and the average volume is shown. d Tumor weights were measured and shown in the graph. e H&E staining of SKOV3 tumors from control mice and mice treated with 5 mg/kg Hu5F9-G4 or different dose of HuNb1-IgG4. Magnification of ×400. The above animal experiments were performed in triplicate and one representative experiment was displayed. Data were presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 versus control

Fig. 4

HuNb1-IgG4 shows low affinity to human RBCs and high safety in cynomolgus monkeys. a The activity of HuNb1-IgG4 and Hu5F9-G4 binding to RBCs isolated form fresh blood of six people. The binding activity was detected by FACS. The binding activity detections from two people were shown. The result from other four people were displayed in Additional file 3. b The effect of HuNb1-IgG4 on RBC agglutination, with Hu5F9-G4 and H6B12 as controls. The experiment was performed in triplicate and one representative experiment was shown. c, d The HGB levels (c) and RBC counts (d) in two individual cynomolgus monkeys administrated with a single intravenous infusion of HuNb1-IgG4 at the doses of 10 mg/kg or 30 mg/kg. e, f The HGB levels (e) and RBC counts (f) in two individual cynomolgus monkeys administrated with a priming dose of 3 mg/kg followed by another dose of 60 mg/kg or 200 mg/kg respectively. The above monkey experiments were performed once

Fig. 5

Large scale production of HuNb1-IgG4 in CHO-S cells expression system. a The viable cell density (VCD) and the viability were detected. b The protein titer was determined within 14 days. c The lactic acid (Lac) and NH4⁺ produced during the culture process. d The thermal stability of HuNb1-IgG4 was detected by SEC-HPLC detection. The experiments were performed in triplicate and one representative experiment was shown

Fig. 6

The anticancer effect of anti-CD47/CD20 bispecific antibody against lymphoma in vivo. a The structure of anti-CD47/CD20 bispecific antibody. b The reductive (left) and nonreductive form (right) of BsAb were displayed through SDS-PAGE. c The purity of BsAb was determined through HPLC analysis. The above experiments were performed in triplicate and one representative experiment was shown. d, e The specificity of CD47/CD20 BsAb towards CD47 (d) or CD20 (e) expressed on Raji cells. Raji cells was co-incubated with HuNb1-biotin or Rituximab-biotin as well as Negative Ab, Rituximab (CD20 mAb), HuNb1-IgG4 (CD47 Nb) or CD47/CD20 BsAb. The binding activity was detected by FACS. f The MFI percentage of Raji cells and RBCs. Raji cells and tenfold excess RBCs were incubated with HuNb1-IgG4 or CD47/CD20 BsAb respectively. FITC positive antibodies binding to cells were detected by FACS. The RBCs were derived from three donors. g CD47/CD20 BsAb induces phagocytosis of target cells. Phagocytosis of CFSE-labeled Raji cells mediated by macrophages was analyzed by FACS. The above experiments were performed in triplicate and data were presented as mean ± SD. **P < 0.01, ***P < 0.001 versus control. h The male NOG mice were injected through tail vein with Raji cells and treated with HuNb1-IgG4, Rituximab or CD47/CD20 BsAb at the same dose (20 mg/kg), with PBS as the control (n = 6). Tumor volumes and the index of tumor volume inhibition (TGI) were shown. The experiment was performed in triplicate and one representative experiment was shown. Data were presented as mean ± SEM. **P < 0.01, ***P < 0.001 versus control