Poster J9 - Design of a single-domain antibody (sdAb)-conjugated photosensitizer and study of its therapeutic activity

Thursday, October 24, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Co-Author(s)

HW

Henan Wang

Graduate student (9/2021-7/2024)
Nankai University, United States

Presenting Author(s)

Zongpei He

Undergraduate Student
Nankai University
Tianjin, Tianjin, China (People's Republic)

Co-Author(s)

SS

Shenyang Shen

Graduate student
Nankai University, United States

Primary Investigator(s)

ZH

Zhangyong Hong

Professor
Nankai University, United States

Introduction: Photodynamic therapy (PDT) is a novel cancer treatment modality based on photosensitizers (PS). The PS can be excited by light at specific wavelengths and generate reactive oxygen species (ROS) within the target tissue, leading to cell death[1]. Antibody-conjugation enhances the tumor specific PDT activity of PS, showing promising clinical applications. However, most antibody-conjugated photosensitizers rely on monoclonal antibodies with large molecular weights. This results in poor permeability into solid tumors, prolonged circulation half-lives in blood that lead to prolonged photosensitivity in patients, and challenges in achieving uniform coupling products due to the complexity of their structures. To address these issues, we developed a single-domain antibody (sdAb)-conjugated photosensitizer.

Single-domain antibodies consist of only one heavy chain and weigh approximately 14 kDa. They serve as promising targeting ligands to develop antibody-conjugated photosensitizer. In vitro and in vivo experiments have demonstrated that sdAb-conjugated photosensitizers can induce tumor specific PDT[2][3].

In our work, 5F7, an sdAb targeting human epidermal growth factor receptor 2 (HER2) was used as the targeting ligand, and the Pyropheophorbidea (Pyro) was used as the photosenistizer. Pyro is stable and easy to prepare, but it is strongly hydrophobic. Therefore, it was pre-connected with a polyethylene glycol (PEG) chain of about 1 kDa to enhance hydrophilicity. The Pyro-PEG1k-NH2 was then site-specifically conjugated to 5F7 sdAb to enhance the uniformity of the final product, NHER2-PEG1k-Pyro. In vitro and in vivo experiments demonstrated that the drug exhibits ideal selectivity and photocytotoxicity against HER2-high expression cells, has a shorter half-life, and shows robust PDT activity.

Materials and

Methods: 1. Characterization of NHER2-PEG1k-Pyro
The purity of 5F7 sdAb protein was assessed by SDS-PAGE. The purity of NHER2-PEG1k-Pyro was assessed by both SDS-PAGE and HPLC.

2. Hydrophilicity analysis
The hydrophilicity of Pyro, Pyro-PEG1k-NH2, and NHER2-PEG1k-Pyro was detected by UV/vis absorption spectra in PBS buffer.

3. In vitro HER2 affinity analysis
The HER2 affinity of 5F7 and NHER2-PEG1k-Pyro were compared using ELISA analysis, with concentration gradient of 5F7 and NHER2-PEG1k-Pyro ranging from 1E-6 to 1E-13.

4. In vitro photocytotoxicity analysis
The photocytotoxicity of Pyro, Pyro-PEG1k-NH2, and NHER2-PEG1k-Pyro were compared using MTT experiments on HER2-high expression cells and HER2-low expression cells.

5. In vivo pharmacokinetic analysis
The distribution of drugs in NCI-N87 tumor mouse model was monitored by in vivo NIR imaging system. And the circulation half-life was detected by collecting serum of mice.

6. In vivo PDT activity of NHER2-PEG1k-Pyro
The in vivo PDT activity of NHER2-PEG1k-Pyro was tested by treating the NCI-N87 tumor mouse model with 20nmol/mouse of drug, using 50-200 mW/cm2 power of light. The mice were monitored for tumor volume and body weight for 40 days.

Results, Conclusions, and Discussions: 1. Characterization of NHER2-PEG1k-Pyro
SDS-PAGE results showed that the purity of 5F7 sdAb protein is excellent. Fluorescence imaging and HPLC both confirmed the high purity of purified conjugated product NHER2-PEG1k-Pyro.

2. Hydrophilicity
UV/vis absorption spectra showed that both Pyro-PEG1k-NH2 and NHER2-PEG1k-Pyro exhibited strong and sharp absorption peaks at 680 nm, suggesting non-aggregation in PBS. However, the absorption spectrum of free Pyro indicating aggregation in PBS because of strong hydrophobicity.

3. In vitro HER2 affinity
ELISA on HER2 receptor showed that the EC50 value of NHER2-PEG1k-Pyro (1.517 nM) was higher than that of 5F7 (1.199 nM) but was still acceptable.

4. In vitro photocytotoxicity
MTT experiments showed that Pyro exhibited very strong photocytotoxicity to both HER2-high expression cells and HER2-low expression cells, while Pyro-PEG1k-NH2 showed lower photocytotoxicity to all cell lines. NHER2-PEG1k-Pyro only showed strong photocytotoxicity to HER2-high expression cells, with an IC50 of 141nM.

5. In vivo pharmacokinetic
In vivo NIR imaging in NCI-N87 mouse model showed that the fluorescence signal of NHER2-PEG1k-Pyro began to accumulate in tumor sites at 2 h, reaching the highest point at 10 h. In contrast, the signal of Pyro-PEG1k-NH2 at tumor sites is weak and decreased rapidly, while Pyro showed almost no signal at tumor sites. These results demonstrated that the conjugation with 5F7 provides Pyro with ideal capability for tumor accumulation. The half-life of NHER2-PEG1k-Pyro in circulation was calculated to be 49.5 min.

6. In vivo PDT activity
HER2-high expression NCI-N87 mouse model was used to detect in vivo PDT activity of NHER2-PEG1k-Pyro. In the treatment group with 20 nmol/mouse of NHER2-PEG1k-Pyro and irradiation with different power of lights (670 nm, 50-200 mW/cm2, 15 min), the tumor finally disappeared in the 200 mW/cm2 subgroup, and no recurrence was observed during 40 days of continuous observation.

Conclusion
In vitro and in vivo experiments showed that NHER2-PEG1k-Pyro has ideal cell selectivity, photocytotoxicity, tumor site accumulation and successfully eliminates HER2-high expression NCI-N87 tumors in a mouse model.

Discussion
The drug still needs a relatively high concentration to eliminate tumor in mouse model, future work can focus on improving the cell-killing ability of the conjugate.

Acknowledgements (Optional): This research constitutes a part of Henan Wang's graduate student project. During my year at Zhangyong Hong's lab, I closely followed this project and took part in the experiments detailed in the Materials and Methods. Before summarizing this research and submitting the abstract, I obtained consent from both Zhangyong Hong and Henan Wang. I would like to express my gratitude to Zhangyong Hong and Henan Wang for providing me with the opportunity to present, and to Shenyang Shen for his participation in this work.

Refreence:
[1] Correia JH, Rodrigues JA, Pimenta S, Dong T, Yang Z. Photodynamic Therapy Review: Principles, Photosensitizers, Applications, and Future Directions. Pharmaceutics. 2021; 13 (9):1332. https://doi.org/10.3390/pharmaceutics13091332.
[2] Raimond Heukers, Paul M.P. van Bergen en Henegouwen, Sabrina Oliveira. Nanobody–photosensitizer conjugates for targeted photodynamic therapy. Nanomedicine: Nanotechnology, Biology and Medicine. 2014; 10 (7):1441-1451. https://doi.org/10.1016/j.nano.2013.12.007.
[3] Pieter B.A.A. van Driel, Martin C. Boonstra, Maxime D. Slooter, Raimond Heukers, Marieke A. Stammes, Thomas J.A. Snoeks, Henriette S. de Bruijn, Paul J. van Diest, Alexander L. Vahrmeijer, Paul M.P. van Bergen en Henegouwen, Cornelis J.H. van de Velde, Clemens W.G.M. Löwik, Dominic J. Robinson, Sabrina Oliveira. EGFR targeted nanobody–photosensitizer conjugates for photodynamic therapy in a pre-clinical model of head and neck cancer. Journal of Controlled Release. 2016, 229: 93-105. https://doi.org/10.1016/j.jconrel.2016.03.014.