Abstract 59: CD4-targeted peptides: Discovery, <i>in vitro</i> characterization, and preclinical PET tracer evaluation in a humanized CD4 knock-in mouse model

Abstract Introduction: Immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment, but their efficacy can vary greatly among patients. This variation may be due to the level of immune infiltration in the tumor microenvironment. Immune surveillance, particularly of tumor-infiltrating leukocytes like CD4+ T-cells, can help predict ICI efficacy. This work presents the discovery and preclinical characterization of two CD4-targeted peptide PET tracers. Methods: Peptide discovery was based on a peptide phage display biopanning, and selected peptides were commercially synthesized (&amp;gt;95% purity). The binding of all peptides to human CD4 (hCD4) was characterized using surface plasmon resonance (SPR) on a Biacore X100. Peptides exhibiting binding to hCD4 were also evaluated for binding to a negative control protein and select peptides to murine CD4 (mCD4). All SPR experiments were analyzed via reference and blank subtraction, and fitted to a 1:1 Langmuir model. The peptides' ability to interact with hCD4 was evaluated using a Sypro Orange thermal shift assay (TSA) on an MxPro3005 qPCR instrument, followed by determination of the midpoint temperature (Tm) via a Boltzmann fit. Peptides 11 (P11) and 17 (P17) underwent DOTA-conjugation (&amp;gt;95% purity) and radiolabeling with copper-64 (64Cu) (&amp;gt;95% radiochemical purity) for in vivo PET/CT imaging of C57BL/6J hCD4 knock-in (KI) and control (wild-type) models on a Siemens Inveon PET/CT scanner. The mice were imaged at 1 and 3 hours post-injection (p.i.), and the PET signal of select organs was decay-corrected and quantified as percentage injected dose per gram (%ID/g). The spleen was the organ of interest due to its relatively high population of CD4+ T-cells. Results: Twenty peptides were synthesized and tested on SPR. P11 and P17 showed selective hCD4 binding on SPR with affinities of KD=0.96 µM and 2.39 µM, respectively. P11 induced a significant Tm shift (57.3±0.28°C, p&amp;lt;0.0001) and [64Cu]Cu-DOTA-P11 PET/CT imaging showed high spleen uptake at 1 and 3 hours p.i. in both models. P11 binding to mCD4 was estimated to KD =26.6 nM. [64Cu]Cu-DOTA-P17 PET/CT imaging showed significantly higher spleen uptake (0.71±0.10 %ID/g, p&amp;lt;0.05) at 1 hour p.i. in the hCD4 KI model, but not at later time-points. Conclusions: P11 bound hCD4 with nanomolar affinity, and a significant Tm increase suggests hCD4-P11 interaction. PET/CT imaging with [64Cu]Cu-DOTA-P11 showed high spleen uptake in both models. This, and P11’s nanomolar affinity towards mCD4 indicates cross-reactivity. For future work [64Cu]Cu-DOTA-P11 needs to be evaluated in a different animal model not reliant on differences between hCD4 and mCD4, e.g., in a high and low expressing hCD4 xenograft flank tumor model. P17 bound hCD4 with low micromolar affinity. PET/CT imaging with [64Cu]Cu-DOTA-P17 showed significantly higher uptake in the hCD4 KI spleen at 1 hour. Citation Format: Anders Wilgaard Sinkjaer, Ida Vang Andersen, Anne Skovsbo Clausen, Line Bruhn Knudsen, Camilla Stavnsbjerg, Andreas Kjaer. CD4-targeted peptides: Discovery, in vitro characterization, and preclinical PET tracer evaluation in a humanized CD4 knock-in mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 59.