Application of 99mTc-Labeled WL12 Peptides as a Tumor PD-L1-Targeted SPECT Imaging Agent: Kit Formulation, Preclinical Evaluation, and Study on the Influence of Coligands

Abstract

With the development of PD-1/PD-L1 immune checkpoint inhibitor therapy, the ability to monitor PD-L1 expression in the tumor microenvironment is important for guiding therapy. This study was performed to develop a novel radiotracer with optimal pharmacokinetic properties to reflect PD-L1 expression in vivo via single-photon emission computed tomography (SPECT) imaging. [^99mTc]Tc-HYNIC-WL12-tricine/M (M = TPPTS, PDA, ISONIC, 4-PSA) complexes with high radiochemical purity (>97%) and suitable molar activity (from 100.5 GBq/μmol to 300 GBq/μmol) were prepared through a kit preparation process. All ^99mTc-labeled HYNIC-WL12 radiotracers displayed good in vitro stability for 4 h. The affinity and specificity of the four radiotracers for PD-L1 were demonstrated both in vitro and in vivo. The results of biodistribution studies displayed that the pharmacokinetics of the ^99mTc-HYNIC-conjugated radiotracers were significantly influenced by the coligands of the radiotracers. Among them, [^99mTc]Tc-HYNIC-WL12-tricine/ISONIC exhibited the optimal pharmacokinetic properties (t_1/2α = 8.55 min, t_1/2β = 54.05 min), including the fastest clearance in nontarget tissues, highest tumor-to-background contrast (e.g., tumor-to-muscle ratio, tumor-to-blood ratio: 40.42 ± 1.59, 14.72 ± 2.77 at 4 h p.i., respectively), and the lowest estimated radiation absorbed dose, highlighting its potential as a clinical SPECT imaging probe for tumor PD-L1 detection.

Keywords: PD-L1; SPECT/CT; Technetium-99m; coligand; peptide WL12.

Scheme 1

Chemical structure of HYNIC-conjugated WL12 (HYNIC-WL12) and coligands (M = TPPTS, ISONIC, PDA, and 4-PSA), and radiolabeling routes of [^99mTc]Tc-HYNIC-WL12-tricine/M complexes.

Figure 1

Radio-HPLC profiles of the stability of [^99mTc]Tc-HYNIC-WL12-tricine/M incubated in saline and serum for 4 h. ((A). M = TPPTS; (B). M = PDA; (C). M = ISONIC; (D). M = 4-PSA).

Figure 2

Cellular uptake of [^99mTc]Tc-HYNIC-WL12-tricine/M in MC38-B7H1 and MC38 cells (A–D) was blocked by WL12 in MC38-B7H1 cells (E–H) (n = 3, ** p < 0.01, *** p < 0.001).

Figure 3

Biodistribution results of [^99mTc]Tc-HYNIC-WL12-tricine/M in MC38-B7H1 (PD-L1-positive)- and MC38 (PD-L1-negative)-bearing mice and blocked results in MC38-B7H1-bearing mice at 2 h p.i. (n = 4, * p < 0.05, ** p < 0.01, *** p < 0.001). (A) Tumor uptake; (B) tumor-to-muscle ratio; and (C) tumor-to-blood ratio.

Figure 4

Metabolic stability of [^99mTc]Tc-HYNIC-WL12-tricine/TPPTS (A–C) and [^99mTc]Tc-HYNIC-WL12-tricine/ISONIC (D–F) in tumor-bearing C57BL/6N mice at 2 h and 4 h p.i.

Figure 5

Whole-body micro-SPECT/CT images of tumor-bearing mice injected with [^99mTc]Tc-HYNIC-WL12-tricine/TPPTS (A,B) or [^99mTc]Tc-HYNIC-WL12-tricine/ISONIC (C,D) at 2 h and 4 h p.i. (A_m = 135~150 GBq/μmol).

Figure 6

Ratios of tumor/muscle, tumor/blood, tumor/lung, and tumor/liver for [^99mTc]Tc-HYNIC-WL12-tricine/M (M = TPPTS, PDA, ISONIC, or 4-PSA, respectively) at 2 h and 4 h p.i.