Background
Antibody-drug conjugates (ADCs) offer advantages such as targeted specificity, potent anti-cancer effects, and a long circulation half-life. However, ADCs also have limitations, such as prolonged systemic exposure, slow clearance, and Fc-mediated side effects. However, drug conjugates based on Fab fragments offer unique features in terms of safety, clearance, and permeability. Current methods for preparing Fab-drug conjugates are limited by the availability and stability of Fab proteins, and reports on this approach are scarce.
On September 2, 2024, Xu Ruolin's research group at the School of Pharmaceutical Sciences, Wuhan University published a research result titled " A single-chain fab derived drug conjugate for HER2 specific delivery " in Biomaterials. The anti-tumor agent SN38 was conjugated to the C-terminus via sortase A ligation, generating a homogeneous Fab conjugate with a drug to Fab ratio of 1. The resulting anti-HER2 Fab-SN38 conjugate was shown to be effective and antigen-dependent, with cell-killing ability due to its special tissue protease-triggered cyclization-promoting release mechanism. In vivo and in vitro experiments showed that the Fab-SN38 conjugate was highly specific for HER2-positive cells and had good therapeutic effects.
scFab structure and its conversion to Fab
The scFab is designed by fusing the C-terminus of the light chain (LC) to the N-terminus of the heavy chain (HC) through a flexible histidine-rich loop. This 63-amino acid loop contains a large number of glycine and serine to maintain its flexibility. The proximity effect of the linker also promotes correct the light/heavy chain pairs and the formation of interchain disulfide bonds both help prevent the "light chain deletion" phenomenon commonly seen in Fab expression.
The histidine-rich loop in the scFab was removed by TEV protease treatment, thereby converting the scFab into a traditional Fab format. The scFab-derived Fab has good targeting and binding capabilities.
Anti-HER2 Fab drug conjugates
To achieve site-specific conjugation with the SN38 payload, the C-terminus of the scFab is encoded with a sortaseA substrate peptide sequence (LPETGG). In the structural design of BCN-VCit-SN38, the payload SN38 is connected to the VCit linker via a glutamate bond. Following internalization by tumor cells, the linker is cleaved by enzymes in the lysosome, triggering a cyclization reaction and achieving traceless release of the payload. BCN-VCit-SN38 reacts with Fab-azide via copper-free click chemistry to form the Fab-SN38 conjugate.
Cathepsin B promotes tandem release of payload
The Fab-SN38 conjugate is likely processed by cathepsins in the acidic endosome, releasing Glu (SN38) -OH. This intermediate must diffuse into the higher pH of the cytoplasm to trigger the ring closure reaction and release the SN38 payload. Glu (SN38) -OH is cell membrane permeable, triggering its cytotoxic activity by releasing the potent SN38.
In vitro efficacy against tumor cells
We observed an additive effect of the Fab-SN38 conjugate on all HER2-positive cell lines after treatment. Fab-SN38 is a potent and selective cytotoxic agent against HER2-positive cells. Both anti-HER2 Fab and SN38 are therapeutic agents targeting HER2-positive tumor cells. Once covalently bound, they exhibited an additive effect on tumor cell growth inhibition. Anti-HER2 Fab-SN38 was labeled with the fluorescent dye sulfo-Cy5 and was found to enter cells via receptor-mediated internalization.
The anti-HER2 Fab arrested the NCI-N87 cell cycle at the G0/G1 phase, which would affect cell growth but not induce apoptosis. In Calu-3 and SK-OV-3 cells, the Fab fragment had no significant effect on the cell cycle. Both Fab-SN38 and its payload SN38 significantly induced cell cycle arrest at the G2/M phase in all three cell lines, suggesting increased apoptosis through DNA damage. This suggests that SN38 plays an indispensable role in anti-tumor activity, particularly in cell lines insensitive to trastuzumab.
In vivo imaging of solid tumor xenografts
Near-infrared (NIR) imaging technology was used to track and monitor the distribution and accumulation of Fab-SN38 in tumor-bearing nude mice. Fab-SN38 accumulated to a lesser extent in the heart, liver, lungs, and spleen, but was primarily observed in tumors. Trastuzumab, on the other hand, was deposited extensively in the liver along with the tumor. Pharmacokinetic analysis showed that the blood circulation time of Fab-SN38 was significantly shorter than that of the full-length antibody.
In vivo therapeutic effects
In vivo, Fab-SN38 prevented the growth of HER2-positive tumors in athymic mice and was well tolerated at a dose of 7 mg/kg.
Summarize
Anti-HER2 Fab-SN38 drug conjugate exhibited excellent specificity and efficacy against HER2- positive tumor cells both in vitro and in vivo through fine-tuning of the conjugation mode and release mechanism. This indicates that antibody fragment-based drug conjugates have potential in targeted tumor chemotherapy and opens up new prospects for the development of novel drug conjugates using Fab.
