Abstract 3111: Preclinical development of a peptide-based immune-surveillance PET tracer for immuno-oncology stratification
Authors: Sinkjaer Anders W. , Sell Danna K. , Dawoodi Ida M. , Bakhshinejad Babak , Stavnsbjerg Camilla , Kjaer Andreas 
Published: March 2024

Abstract
Introduction: Cancer immunotherapies, such as immune checkpoint inhibitors (ICIs), have become an integral part of cancer treatment. However, significant differences in their effectiveness persist among individual patients. This variability partly stems from poorly immunogenic tumors, referred to as "cold" tumors, characterized by low levels of tumor-infiltrating leukocytes (TILs). Studies indicate that lower levels of TILs, specifically CD4+ and CD8+ T-cells, lead to reduced efficacy of certain immunotherapies. Therefore, the imaging of these cell types could be invaluable in screening cancer patients to predict the effectiveness of a prescribed immunotherapeutic agent before and during treatment. CD4+ T-cells, identified by their high expression of CD4 receptors, could serve as a potential molecular imaging target for an immuno-oncology relevant PET tracer. For this purpose a peptide-based PET tracer offer several beneficial characteristics such as relatively low circulation time, cell penetration, and low development costs.
Methods: All peptides were identified through peptide phage display (PPD) and commercially synthesized (&gt;95% purity). These peptides underwent screening for their binding affinity to recombinant human CD4 and a negative control protein using surface plasmon resonance (SPR) on a Biacore X100. Peptides exhibiting specific binding to CD4, as determined by SPR, were further selected for FITC-labeling (&gt;95% purity). FITC-labeled peptides were applied in fluorescence-based cell assays conducted on the SUP-T1 (CRL-1942) cell line and an established SUP-T1 CD4 knockout (KO) cell line. Flow cytometry assays were performed using a Fortessa X-20, while live cell imaging was conducted on either a LSM980 or LSM900 confocal microscopy. Simultaneously, an in silico docking method was employed, utilizing ColabFold—an online complex predictor powered by AlphaFold2—for CD4-peptide docking analysis.
Results: Several peptides successfully identified via PPD exhibited binding to recombinant human CD4 as shown by SPR screening. The FITC-labeling and assessment against SUP-T1 and corresponding CD4 KO cell line unveiled specific CD4 binding within a subset of peptides, as observed in both flow cytometry and confocal microscopy. In vitro data was compared to CD4-peptide docking results to create a list of peptides for future in vivo studies.
Conclusions: Multiple peptides successfully identified via PPD showed robust in vitro CD4 binding through SPR screening. Select peptides also demonstrated specific CD4 binding in cell assays, corroborated by docking results. Future prospective for the high performing peptides include in vivo PET/CT Ga-68 or Cu-64 imaging in tumors with high and low CD4+ T-cell infiltration.
Citation Format: Anders W. Sinkjaer, Danna K. Sell, Ida M. Dawoodi, Babak Bakhshinejad, Camilla Stavnsbjerg, Andreas Kjaer. Preclinical development of a peptide-based immune-surveillance PET tracer for immuno-oncology stratification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3111.