Imaging of Fibroblast Activation Protein in Cancer Xenografts Using Novel (4-Quinolinoyl)-glycyl-2-cyanopyrrolidine-Based Small Molecules

Abstract

Fibroblast activation protein (FAP) has become a favored target for imaging and therapy of malignancy. We have synthesized and characterized two new (4-quinolinoyl)-glycyl-2-cyanopyrrolidine-based small molecules for imaging of FAP, QCP01 and [¹¹¹In]QCP02, using optical and single-photon computed tomography/CT, respectively. Binding of imaging agents to FAP was assessed in six human cancer cell lines of different cancer types: glioblastoma (U87), melanoma (SKMEL24), prostate (PC3), NSCLC (NCIH2228), colorectal carcinoma (HCT116), and lung squamous cell carcinoma (NCIH226). Mouse xenograft models were developed with FAP-positive U87 and FAP-negative PC3 cells to test pharmacokinetics and binding specificity in vivo. QCP01 and [¹¹¹In]QCP02 demonstrated nanomolar inhibition of FAP at K_i values of 1.26 and 16.20 nM, respectively. Both were selective for FAP over DPP-IV, a related serine protease. Both enabled imaging of FAP-expressing tumors specifically in vivo. [¹¹¹In]QCP02 showed high uptake at 18.2 percent injected dose per gram in the U87 tumor at 30 min post-administration.

Figure 1.

FAP expression in human cancers. FAP mRNA expression in human cancer cell lines from the CCLE (A) and in human primary tumors from TCGA (B) was analyzed. FAP mRNA expression in primary tumors and normal tissues from TCGA data was also analyzed (C); NSCLC = non-small-cell lung cancer; SCLC = small-cell lung cancer; DLBCL = diffuse large B-cell lymphoma; AML = acute myeloid leukemia; CML = chronic myelogenous leukemia; PAAD = pancreatic cancer; BRCA = breast cancer; MESO = mesothelioma; HNSC = head and neck cancer; LUAD = lung adenocarcinoma; LUSC = lung squamous cell carcinoma; STAD = stomach cancer; UCS = uterine carcinosarcoma; ESCA = esophageal cancer; READ = rectal cancer; DLBC = large-B-cell lymphoma; COAD = colon cancer; SKCM = melanoma; CHOL = bile duct cancer; BLCA = bladder cancer; CESC = cervical cancer; UCEC = endometrioid cancer; OV = ovarian cancer; KIRC = kidney clear cell carcinoma; TGCT = testicular cancer; THCA = thyroid cancer; GBM = glioblastoma; PRAD = prostate cancer; ACC = adrenocortical cancer; PCPG = pheochomocytoma; LIHC = liver cancer; LGG = lower-grade glioma; KIRP = kidney papillary cell carcinoma; UVM = ocular melanomas; KICH = kidney chromophobe; THYM = thymoma; LAML = acute myeloid leukemia.

Figure 2.

In vitro binding and specificity of QCP01 and [¹¹¹In]QCP02. (A) Cells incubated with various concentrations (range: 50–0.78 nM) of QCP01 were imaged with the LI-COR Pearl Impulse Imager to assess binding of the agent in various FAP-positive (+) and FAP-negative (−) cell lines (left). Dose–response curves of QCP01 binding in FAP-positive cell lines (NCIH2228, U87, and SKMEL24) and FAP-negative cell lines (PC3, NCIH226, and HCT116) were generated (right). (B) Cells were incubated with 0.037 MBq [¹¹¹In]QCP02 and were washed with cold phosphate-buffered saline (PBS). The radioactivity of the cell pellets was measured and normalized to the incubated dose; ****, P < 0.0001. (C) Cells incubated with 25 nM QCP01 were incubated with various concentrations of either a DPP-IV and FAP inhibitor, Val-boroPro, or a DPPIV-only inhibitor, sitagliptin. The binding of QCP01 was measured, and semi-log inhibitor–response curves were generated for both Val-boroPro (left) and sitagliptin (right).

Figure 3.

NIRF imaging of QCP01 in a tumor-bearing mouse. NOD/SCID mice bearing FAP-positive U87 (red) and FAP-negative PC3 (white) tumor xenografts (n = 4) were injected with 5 nmol of QCP01 via the tail vein, followed by serial NIRF imaging on the LI-COR Pearl Impulse Imager. Representative images of QCP01 full body (left) distribution at 5 h after injection and organ-specific (right) distribution at 5, 24, and 48 h after injection are shown.

Figure 4.

Serial SPECT-CT imaging of [¹¹¹In]QCP02 in a tumor-bearing mouse. A NOD/SCID mouse bearing FAP-positive U87 (red) and FAP-negative PC3 (blue) tumor xenografts was injected with 7.4 MBq [¹¹¹In]QCP02 via the tail vein, followed by serial SPECT-CT imaging. Representative three-dimensional SPECT-CT images at various time points after injection (1, 3, 6, 10, and 28 h) are shown.

Scheme 1.. Chemical Synthesis of QCP01 and QCP02^a

^aReagents and conditions: (a) glycine methyl ester hydrochloride, HOBt, HBTU, ⁱPr₂NEt, DMF, RT, 6 h, 76%; (b) 3-(Boc-amino) propyl bromide, Cs₂CO₃, DMF, RT, 16 h, 54%; (c) LiOH, H₂O/THF, RT, 6 h, 99%; (d) (S)-pyrrolidine-2-carbonitrile hydrochloride, HOBt, HBTU, ⁱPr₂NEt, DMF, RT, 6 h, 80%; (e) TFA/CH₂Cl₂, RT, 1 h; (f) IRDye 800CW-NHS ester, ⁱPr₂NEt, DMF, RT, 2 h, 81% (total yield e and f); (g) 1. DOTA-GA(t-Bu)₄-NHS, ⁱPr₂NEt, DMF, RT, 4 h, 2. TFA/H₂O/TES, RT, 1 h, 35%.

Chart 1.

FAP Inhibitors