Poster J18 - Synthesis of Protein Degrading IgG-CXCL12 Compound

Saturday, October 26, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Andrew C. Chiang

Undergraduate Student
University of Pennsylvania
Philadelphia, Pennsylvania, United States

Primary Investigator(s)

Andrew Tsourkas, PhD

Primary Investigator
University of Pennsylvania
Philadelphia, Pennsylvania, United States

Introduction: Targeted protein degradation (TPD) is a promising approach for treating many diseases, such as cancer, because it allows for the removal of proteins that are responsible for driving disease progression. There are many proteins that can be dysregulated in disease, including many surface proteins. Several methods have utilized lysosomal degradation to selectively eliminate target proteins on the cell surface. One method to eliminate target proteins on the cell surface is cytokine receptor-targeting chimeras (KineTACs) [1]. KineTACs are modified antibodies with cytokine, such as CXCL12, attached to it.

Figure 1a illustrates how KineTACs works. In the first step, the IgG-CXCL12 conjugate binds to its target protein on the cell surface while the CXCL12 portion of the conjugate binds to its corresponding receptor CXCR7 on the cell membrane. In the second step, the binding of the CXCL12 receptor induces endocytosis. In the third step, the vesicle reaches and merges with the lysosome. Subsequently, the lysosomal enzymes degrade everything inside the lysosome.

KineTACs are currently developed by genetically engineering antibodies with CXCL12 fused to the n-terminus of the heavy chain. Therefore, for every new target protein, a new antibody must be engineered. We present an efficient and simple approach to attach CXCL12 to any off-the-shelf IgG using photoreactive antibody-binding domains (pAbBDs), where two pAbBD-CXCL12 fusion proteins are attached to each antibody (Figure 1b). The process of attaching the fusion proteins to the antibody only takes two hours, and can be parallelized, allowing tens to hundreds of antibody-CXCL12 conjugates to be produced at once.

Materials and

Methods: To enable attachment of CXCL12 to IgG, we generated proteins that have photoreactive antibody-binding domains (pAbBDs) fused with the desired cytokine, CXCL12. The pAbBD contains an unnatural amino acid (benzoylphenylalanine, or BPA) which allows the pAbBD to be covalently linked to the heavy chains of any IgG upon exposure to ultraviolet light [2].

The pAbBD-CXCL12 fusion protein is expressed in E. coli. To extract the protein, the E. coli cells were first lysed in non-denaturing solution and sonicated. The pellet is extracted after centrifugation. The pellet is resuspended in 7M urea. The urea is extracted through dialysis in 1X PBS for 24 hours. We attached an additional sortase A (SrtA) tag in the pAbBD-CXCL12 conjugate to enable the usage of the Sortase-Tag Expressed Protein Ligation (STEPL) system to purify pAbBD-CXCL12 [3]. The his-tag is captured on cobalt beads and the SrtA enzyme releases the pAbBD-CXCL12 from the beads upon addition of triglycine. IgG beads are then used to isolate properly folded pAbBD-CXCL12 conjugates.

The pAbBD-CXCL12 was attached to an off-the-shelf anti-EGFR antibody Cetuximab through photo-crosslinking. Since each IgG has two heavy chains, a maximum of two pAbBD can be crosslinked to an IgG.

Cell studies were performed with 3 groups: cells only (control), Cetuximab only, and Cetuximab-CXCL12 conjugates. A431 cells were used for all studies. The treatments were applied to A431 cells for 24 hours at a dose of 100 nM and 37 °C. The level of EGFR following treatment was determined through semi-quantitative western blot.

Results, Conclusions, and Discussions: Figure 2 shows the SDS-PAGE of Cetuximab alone or following photocrosslinking with the pAbBD-CXCL12. Since the pAbBD-CXCL12 is crosslinked to the constant region of the heavy chain, the crosslinked product shows a distinct upward shift by SDS-PAGE. As shown, approximately 90% of the IgG heavy chain is labeled.

The ability of cetuximab-CXCL12 to degrade EGFR was determined through a cell study using A431 cells, where the degradation of EGFR was measured after 24 hours of incubation. Cells were treated with 100 nM of cetuximab or cetuximab-CXCL12 conjugates. Figure 3 shows the extent of EGFR degradation through a semi-quantitative western blot using the LI-COR Odyssey system, where GAPDH was used as the housekeeping control protein. As shown, cetuximab-CXCL12 led to 44% degradation of EGFR compared with controls.

Current methods to create KineTACs require creating new recombinant antibodies. In other words, whenever a new target protein is selected, a new antibody-CXCL12 fusion protein needs to be genetically engineered and expressed. Genetic methods require at least several days to weeks to create bispecific antibodies and the antibody sequence must be known [1]. For our approach, it takes 2 hours to conjugate pAbBD-CXCL12 to the heavy chain of any off-the-shelf antibody, with no prior knowledge of antibody sequence required. Therefore, pAbBD-CXCL12 fusion proteins provide a versatile tool to rapidly generate antibody conjugates to study the targeted degradation of cell surface proteins.

Reference:
[1] Pance, Katarina, et al. https://doi.org/10.1038/s41587-022-01456-2
[2] Zappala, Fabiana, et al. https://doi.org/10.1007/978-1-4939-9654-4_18
[3] Warden-Rothman, Robert, et al. https://doi.org/10.1021%2Fac402871k

Acknowledgements (Optional):