Background

Bispecific antibodies are artificial molecules that fuse the two distinct antigen-binding sites of a monoclonal antibody into a single entity. They have emerged as a promising next-generation anticancer therapy. Despite their fascinating applications, their design and production remain cumbersome and challenging, resulting in a lengthy R&D process and high production costs.

On April 30, 2024, researchers published a study titled "Macrophage-engaging peptidic bispecific antibodies (pBsAbs) for immunotherapy via a facile bioconjugation strategy." The study reported an unprecedented strategy for conjugating tumor-targeting cyclic peptides to the surface of monoclonal antibodies, creating a novel bispecific antibody, peptide bispecific antibodies (pBsAbs). This design combines the advantages of highly specific monoclonal antibodies and serum-stable cyclic peptides, endowing monoclonal antibodies with the additional tumor-targeting ability of binding to two different antigens.

Overview of Novel Peptide Bispecific Antibodies

Bispecific antibodies can recognize two or more different epitopes or antigens on a single or multiple target cells. Bispecific antibodies have two main mechanisms of action, one of which is to recruit immune cells to cancer cells: bispecific antibodies can use one arm to bind to a tumor-specific antigen and the other arm to bind to an immune cell marker (such as CD3) on T cells. In this way, bispecific antibodies can effectively bring immune cells into direct contact with cancer cells, promoting their destruction. Another mechanism involves simultaneously blocking two signaling pathways on target cells, providing a synergistic therapeutic effect by inhibiting multiple disease-related targets. This dual binding ability of bispecific antibodies has opened up avenues for innovative treatments in areas such as oncology and autoimmune diseases.

(Data source: Labrijn AF, et al. Nat Rev Drug Discov. 2019)

Bispecific antibodies can be divided into IgG-like and non-IgG-like structures. The IgG-like subtype maintains the traditional IgG basic structure, containing two Fab arms and one Fc region. This structure has a longer half-life and stability, similar to natural IgG antibodies, and enables them to effectively engage the immune system. Non-IgG-like subtypes are generally smaller and lack the Fc region, which may result in a shorter half-life but may improve tissue penetration. pBsAbs are a new type of peptide bispecific antibody composed of an EGFR-binding cyclic peptide and an anti-SIRP-α monoclonal antibody.

pBsAb design and targeting mechanism

Peptide bispecific antibodies ( pBsAbs ) consist of a monoclonal antibody covalently conjugated to a tumor-targeting cyclic peptide. Linear EBP is first cyclized via a bifunctional linker to form a cyclic EBP (cEBP-OPA). SIRP-α (CD172a) is a macrophage receptor that negatively controls cytotoxic effector functions. When SIRP-α on macrophages binds to CD47 on cancer cells, a "don't eat me" signal is activated, inhibiting macrophage-mediated phagocytosis of cancer cells. EGFR is overexpressed in many cancer cells. To bridge the gap between cancer cells and macrophages, a cyclic EPB was conjugated to an anti-SIRP-α monoclonal antibody to create a novel peptide bispecific antibody (pBsAb). Cyclic peptides are highly stable in serum and are expected to resist enzymatic degradation, significantly extending the molecule's half-life.

The EBP in pBsAbs can target EGFR overexpressed on the surface of cancer cells, while the anti-SIRP-α monoclonal antibody is used to target SIRP-α on macrophages. The pBsAbs bispecific antibody can enhance the interaction between macrophages and cancer cells and block the "don't eat me" checkpoint signal between CD47-SIRP-α, thereby preventing the inactivation of CD47-mediated phagocytosis of cancer cells by macrophages and promoting antibody-dependent cellular phagocytosis (ADCP) and 3D cell spheroid infiltration.

pBsAb proof-of-concept

ELISA assays revealed that pBsAb bound to EGFR in a dose-dependent manner, whereas the monoclonal antibody showed no binding activity to EGFR. Compared to the native mAb, chemical modification of the pBsAb did not significantly interfere with its SIRP-α binding ability.

Further in vitro target binding assays using confocal microscopy and flow cytometry revealed that both the anti-SIRP-α mAb and the pBsAb bound to SIRP-α on the surface of SIRP-α -positive RAW264.7 mouse macrophages. Regarding EGFR binding, fluorescent signals were only observed on EGFR-positive A549 and HT29 cells, but not on EGFR-negative HeLa cells, to which the anti-SIRP-α mAb was unable to bind. This demonstrates that the novel pBsAb can selectively bind to both EGFR and SIRP-α proteins on the cell surface, confirming that, in vitro, this antibody can acquire additional targeting capabilities against EGFR on cancer cells using this novel approach.

A greater number of macrophages were detected in the presence of pBsAb, and macrophage-cancer cell adhesion was enhanced compared with the mAb and PBS negative controls.

Further investigation of its antibody-dependent cellular phagocytosis (ADCP) activity by confocal microscopy and cytometry revealed that phagocytosis occurred in RAW264.7 macrophages, and incubation with an anti-SIRP-α mAb resulted in a significant decrease in phagocytic activity. Using flow cytometry to quantify ADCP activity, the percentage of double-positive phagocytic macrophages increased by 2.0-and 2.5-fold, respectively, in the presence of pBsAb compared to mAb control treatment, targeting EGFR-positive A549 and HT29 cells. However, for EGFR-negative HeLa cells, treatment with either pBsAb or mAb showed similar percentages of phagocytic macrophages, indicating that no enhancement of phagocytic activity was observed. These results suggest that the novel EGFR×SIRP-α pBsAb enhances ADCP activity.

in a three-dimensional HT29 spheroid model revealed that the number of macrophages (green) within the spheroid core treated with pBsAb was significantly higher than that treated with anti-SIRP-α mAb. In the presence of pBsAb, macrophages can effectively bind to and infiltrate the solid tumor core, thereby enhancing the anti-cancer effect.

Summary

In this study, a novel and powerful chemical method was developed to generate a new type of bispecific antibody, namely a peptide bispecific antibody (pBsAb), from a monoclonal antibody. The EGFR×SIRP-α pBsAb was derived from an anti-SIRP-α monoclonal antibody by conjugating an EGFR-targeting cyclic peptide to the protein surface using a powerful peptide cyclization and antibody conjugation reaction. The results showed that the pBsAb was able to bind to EGFR-overexpressing cancer cells and SIRP-α-expressing macrophages, thereby initiating macrophage-cancer cell interactions and enhancing EGFR-targeting antibody-dependent cellular phagocytosis. The successful establishment of this novel platform provides a means to rapidly produce bispecific antibodies for immunotherapy at a lower cost.