Discovery of a common light chain bispecific antibody targeting PD-1 and PD-L1 by Hybridoma-to-Phage-to-Yeast (H2PtY) platform

Abstract

Background: Therapeutic antibody drugs targeting the PD-1 pathway are generally characterized by relatively low response rates and susceptibility to drug resistance during clinical application. Therefore, there is an urgent need for alternative therapeutic strategies to increase the immune response rate. Bispecific antibodies co-targeting PD-1 and PD-L1 may have greater potential to improve the efficacy of the immune checkpoint pathway.

Method: In this study, we developed a potent humanized common light chain (CLC) IgG shape bispecific antibody (bsAb), named JMB2005, based on Hybridoma-to-Phage-to-Yeast platform. The platform allowed us to discover CLC bsAb from traditional mice for any pair of given targets.

Results: JMB2005 exhibited favorable developability, good manufacturing property, and satisfactory efficacy, which could be given via subcutaneous injection at the concentration of 120 mg/mL. Mechanistically, JMB2005 could bridge tumor cells and T cells with both Fab arms and promote T-cells to function as direct tumor cell killers. It could also promote T cell activation by blocking the binding of PD-L1 to CD80. Furthermore, JMB2005 has exhibited a favorable half-life and has demonstrated promising anti-tumor therapeutic efficacy in vivo.

Conclusion: Consequently, the present study showed that the novel humanized CLC bsAb JMB2005 may represent a novel therapeutic agent of great clinical potential.

Keywords: JMB2005; PD-1; PD-L1; bispecific antibody; common light chain.

Figure 1

Identification of CLC bsAbs with an H2PtY platform. We first subjected mouse to PD-1 immunization, and subjected rat to PD-L1 immunization. Hybridoma cells were obtained and then antibody engineering and humanization were applied to obtain the humanized anti-PD-1 Ab. In another module, VH genes from immunized rats were combined with the light chain from the humanized anti-PD-1 mAbs in phage display library, the scFv library was screened, and then transferred to yeast surface. FACS sorting selected VH genes were humanized and anti-PD-L1 mAbs were identified. Humanized anti-PD-1 and anti-PD-L1 mAbs share the same light chain were assembled to CLC bsAbs candidates, knob-into-hole mutation was inserted into Fc region.

Figure 2

Degradation of bsAb candidates. (a) Change in %monomer measured by SEC-HPLC at 40°C for 4 weeks. (b) Change in %IgG purity measured by nrCE-SDS at 40°C for 4 weeks. (c) Change in % acidic peak, main peak, and basic peak measured by iCIEF at 40°C for 4 weeks.

Figure 3

Bioactivity of JMB2005 in vitro. (a) Binding affinity to human PD-1 by BLI. (b) Binding affinity to human PD-L1 by BLI. (c) Cell bridging assay by flow cytometry. (d) MLR of allogeneic immature DCs and CD4 T cells in the presence of test antibodies as indicated. IL-2 level in the culture media was determined by ELISA. (e) Binding activity to Jurkat PD-1-NFAT-luc T cells. (f) Binding activity to PD-L1/CHO aAPC cells. (g) Inhibition of PD-1/PD-L1 pathway.

Figure 4

Antitumor efficacy and PK study of JMB2005 in vivo. (a) In A375 human tumor xenograft mouse model, 40 tumor-bearing mice were divided into 5 groups. Statistically significant tumor volume reduction by JMB2005 at a dose of 5 mg/kg compared to the isotype control group. (b) In CT-26-hPDL1 murine colon cancer model, 40 Balb/c-hPD1/hPDL1 knock-in mice-bearing CT-26-hPDL1 tumors were divided into 5 groups and subjected to their respective treatment regimens. JMB2005, administered at a dosage of 5 mg/kg, demonstrated a statistically significant reduction in tumor volume relative to the isotype control group. (^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001 vs isotype control group). (c) Pharmacokinetics detection of JMB2005 in hFcRn transgenic mice.

Figure 5

Stability evaluation of JMB2005 at 120 mg/mL. (a) Change in % monomer measured by SEC-HPLC at 5°C, 25°C and 40°C for 28 days. (b) Change in %IgG measured by CE-SDS at 5°C, 25°C and 40°C for 28 days. (c) Change in % acidic peak, main peak and basic peak measured by iCIEF at 5°C, 25°C and 40°C for 28 days. (d) Viscosity measurement at 0 mg/mL, 60 mg/mL, 100 mg/mL and 120 mg/mL.