Shanghai Cellular Therapy Group Enhances Antitumor Efficacy with LNP-Delivered mRNA Encoding PD-1/CTLA-4 Bispecific Nanobody

Jun 20, 2024 18:00 CST Updated 18:00

Shanghai Cell Therapy Group

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Immune Checkpoint Inhibitors（ICI）Significant breakthroughs have been made in the field of immuno-oncology.PD-1/PD-L1 and CTLA-4 inhibitors have shown significant efficacy in the treatment of various cancers., especially in clinical trials of metastatic melanoma, the combination of anti-PD-1 and anti-CTLA-4 can increase response rates by up to 60%.

However, this treatment is often accompanied by side effects, which are unbearable for some patients.ICI-related Immune-related Adverse Events（irAE）Related to Fc receptors in immune cells。

Akeso Biopharma'sAK104is a type ofFc Inactive ConfigurationTetravalent bispecific antibody, has been approved by the NMPA for the treatment of advanced cervical cancer.

In a clinical trial targeting advanced gastric or gastroesophageal junction adenocarcinoma, patients treated with AK104 in combination with chemotherapy demonstrated 65.9%Objective Response Rate（ORR）MEDI5752 is a bispecific monoclonal antibody targeting PD-1/CTLA-4 developed by AstraZeneca, which is currently undergoing multiple clinical trials.

Despite these advances, antibody-based therapies still face challenges, including uneven distribution within tumors, prolonged serum half-life, and immunogenicity.

NanobodyWith advantages such as small size, high stability, strong antigen-binding affinity, good water solubility, and natural origin, they have become attractive reagents for developing innovative therapeutic strategies.

Moreover, the limitations of bispecific antibodies can be addressed through in vitro transcribed mRNA, which facilitates the continuous endogenous production of antibodies, and LNPs are currently widely used as mRNA delivery formulations.

On May 28, 2024, researchers from the Medical College of Shanghai University and the Shanghai Cell Therapy Group published a research article in the Nature sub-journal 'Oncogene',Developed a targetedZ15-0, a bispecific nanobody targeting PD-1 and CTLA-4，And optimized the mRNA sequence of coding Z15-0, obtaining Z15-0-2.。

Through in vitro and in vivo experiments, it was confirmed that the Z15-0-2 mRNA sequence significantly enhanced the expression of bispecific nanobodies. Particularly, in a mouse colon cancer model, Z15-0-2 demonstrated better anti-tumor effects.

Specifically, Z15-0 was constructed by connecting the PD-1 and CTLA-4 nanobodies from alpacas using a G4S linker.To enhance its stability and half-life while reducing its immunogenicity, the researchers incorporated IgG4 Fc.。

Through Surface Plasmon Resonance（SPR）Technical analysis of the binding affinity of Z15-0 for PD-1 and CTLA-4,The KD values generated were 675 pM and 3150 pM, respectively., and evaluated the binding ability of Z15-0 to the cell surface by flow cytometry.(Figure 1)。

Figure 1 Structure and Characteristics of Z15-0 Nanobody

Subsequently, theyCode Qα（QPRFAAA）The Exin21 cis-regulatory motif has been integrated into the Z15-0 sequence.，Thereby forming Z15-0-2（CN202311358648.1）。

Figure 2 shows the mRNA structure of Z15-0-2 and the arrangement of bispecific nanobodies. The incorporation of the Exin21 sequence can promote protein synthesis by enhancing mRNA stability.

Figure 2 Structure and Characteristics of Z15-0-2 Antibody

Subsequently, they established an MC38 tumor model in hCTLA-4/hPD-1 transgenic C57BL/6 mice to evaluate the inhibitory effects of Z15-0 and Z15-0-2 on tumor growth.

The results showed,Z15-0 and Z15-0-2 at a dose of 0.5 mg/kg both showed better inhibitory effects on tumor growth compared to the control group and AK104 approved by Akeso Biopharma.(Figure 3)。

Figure 3 Z15-0 and Z15-0-2 Show Comparable Antitumor Efficacy in hCTLA-4/hPD-1 Transgenic C57BL/6 Mouse Colon Cancer Model

Subsequently, through electroporation and LNP transfection of CHO cells, it was found that the expression level of Z15-0-2 mRNA was significantly higher than that of Z15-0 mRNA. Additionally, by injecting Z15-0 or Z15-0-2 mRNA in mice, it was observed that the secretion level of bispecific nanobodies in the Z15-0-2 group was significantly higher than that in the Z15-0 group at the same dose.

Figure 4 Insertion of Qα leads to increased expression of Z15-0-2 mRNA in vitro and in vivo

Through mixed lymphocyte reaction（MLR）In the experiment, the researchers evaluated the immunomodulatory activity of Z15-0 and Z15-0-2 LNP-mRNA.

In brief, PBMCs from two healthy donors were used to isolate CD4 T cells and DCs, respectively. The mRNA-LNP encoding the antibody was first co-incubated with DCs for 24 hours, followed by co-culture with CD4 T cells for 5 days. The supernatant was then collected, and IL-2 release was measured.

Figure 5 Efficacy of Z15-0 and Z15-0-2 LNP-mRNA In Vitro and In Vivo

The results showed,Z15-0-2 LNP-mRNA Can Significantly Increase the Secretion of IL-2。In in vivo tumor models, Z15-0-2 LNP-mRNA showed a higher tumor growth inhibition rate than Z15-0 LNP-mRNA at a dose of 0.42 mg/kg.。

Subsequently, they collected tumor tissues from different dose groups of Z15-0-2 LNP-mRNA treatment, and analyzed the antibody concentration, cytokine concentration, and immune cell populations in the tumors.

Figure 6: Z15-0-2 exerts its effects by remodeling the tumor microenvironment

The results showed that Z15-0-2 LNP-mRNA exerted antitumor effects by regulating cytokine levels and immune cell population composition in the TME.

Conclusion

In summary, this study developed a unique bispecific nanobody, Z15-0, capable of simultaneously targeting PD-1 and CTLA-4. Additionally, this antibody can be delivered via LNP-mRNA.（Z15-0-2）Delivery to achieve effective expression of bispecific antibodies, and significantly reduced tumor growth in a mouse model of colon cancer. Therefore, as an innovative bispecific nanobody for cancer immunotherapy, Z15-0-2 shows great potential.

Original link:

https://www.nature.com/articles/s41388-024-03066-5#Sec10

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