Targeted Imaging of the Atypical Chemokine Receptor 3 (ACKR3/CXCR7) in Human Cancer Xenografts

Abstract

The atypical chemokine receptor ACKR3 (formerly CXCR7), overexpressed in various cancers compared to normal tissues, plays a pivotal role in adhesion, angiogenesis, tumorigenesis, metastasis and tumor cell survival. ACKR3 modulates the tumor microenvironment and regulates tumor growth. The therapeutic potential of ACKR3 has also been demonstrated in various murine models of human cancer. Literature findings underscore the importance of ACKR3 in disease progression and suggest it as an important diagnostic maker for non-invasive imaging of ACKR3 overexpressing malignancies. There are currently no reports on direct receptor-specific detection of ACKR3 expression. Here we report the evaluation of a radiolabeled ACKR3-targeted monoclonal antibody (ACKR3-mAb) for the non-invasive in vivo nuclear imaging of ACKR3 expression in human breast, lung and esophageal squamous cell carcinoma cancer xenografts. Methods ACKR3 transcripts were extracted from Cancer Cell Line Encyclopedia (CCLE), The Cancer Genome Atlas (TCGA) and the Clinical Lung Cancer Genome Project (CLCGP). 89Zr-ACKR3-mAb was evaluated in vitro and subsequently in vivo by positron emission tomography (PET) and ex vivo biodistribution studies in mice xenografted with breast (MDA-MB-231-ACKR3 (231-AC-KR3), MDA-MB-231 (231), MCF7), lung (HCC95) or esophageal (KYSE520) cancer cells. In addition, ACKR3-mAb was radiolabeled with Iodine-125 and evaluated by single photon emission computed tomography (SPECT) imaging and ex vivo biodistribution studies. Results ACKR3 transcript levels were highest in lung squamous cell carcinoma (LUSC) among the 21 cancer type data extracted from TCGA. Also, CLCGP data showed that LUSC has the highest CXCR7 transcript levels compared to other lung cancer subtypes. The 89Zr-ACKR3-mAb was produced in 80±5% radiochemical yields with >98% radiochemical purity. In vitro cell uptake of 89Zr-ACKR3-mAb correlated with gradient levels of cell surface ACKR3 expression observed by flow cytometry. In vivo PET imaging and ex vivo biodistribution studies in mice with breast, lung and esophageal cancer xenografts consistently showed enhanced 89Zr-ACKR3-mAb uptake in high ACKR3 expressing tumors. SPECT imaging of 125I-ACKR3-mAb showed the versatility of ACKR3-mAb for in vivo monitoring of ACKR3 expression. Conclusions Data from this study suggest ACKR3 to be a viable diagnostic marker and demonstrate the utility of radiolabeled ACKR3-mAb for in vivo visualization of ACKR3 overexpressing malignancies.

Antibodies Targeting Chemokine Receptors CXCR4 and ACKR3

“…To our knowledge, currently there are no reported therapeutic mAbs against ACKR3 in preclinical or clinical development. One group reported an application of 89 Zr-11G8, a radiolabeled murine IgG1, for positron emission tomography studies in xenograft mice but without any therapeutic effects described (Behnam Azad et al, 2016). This contrasts with a relatively broad panel of mAbs targeting CXCR4, as summarized in Table 2.…”

“…mAbs targeting CXCR4 or ACKR3 were used as in vivo imaging tools. In this case, antibodies were radiolabeled with 89 Zr and could detect GPCR expression in xenograft tumors in mice using positron emission tomography (Azad et al, 2016;Behnam Azad et al, 2016). To our knowledge, no GPCRtargeting nanobodies have been used thus far for in vivo imaging.…”

Modulators of CXCR4 and CXCR7/ACKR3 Function

“…CXCL12 has been labeled using an iodine-125 label on tyrosine residues and also with Alexa Fluor 647 (Bleul et al, 1996;Oberlin et al, 1996;Hesselgesser et al, 1997Hesselgesser et al, , 1998Di Salvo et al, 2000). Radioiodination has also been introduced to CXCR4 and ACKR3 antibodies and peptides (Endres et al, 1996;Koglin et al, 2006;Han et al, 2010;Demmer et al, 2011a;Behnam Azad et al, 2016). Furthermore, small molecules have been labeled using an array of radiolabels with different tracers ( 18 F, 11 C, 64 Cu, 68 Ga, 89 Zr, or 177 Lu) either as substitutes of unlabeled atoms 748 Adlere et al at ASPET Journals on August 11, 2020 molpharm.aspetjournals.org Downloaded from or in a chelate complex.…”

“…Nowadays, PET is a detection method used both in clinics and research and PET tracers are useful tools in theranostics (Yordanova et al, 2017). 89 Zr-labeled ACKR3-mAb (clone 11G8) was evaluated in vitro and in vivo by PET, which showed enhanced labeled-antibody uptake in high-ACKR3-expressing tumors, and serves as a viable diagnostic marker (Behnam Azad et al, 2016).…”

Chemokines and chemokine receptors: A new strategy for breast cancer therapy

“…It was found that the expression of ACKR3 at protein and mRNA levels in precancerous breast cancer was much higher than that in adjacent and normal breast tissues 26 . Several studies have shown that the high expression of ACKR3 is positively correlated with the proliferation of breast cancer cells, indicating that ACKR3 has a positive effect on tumor growth 27‐29 . Furthermore, Boudot and his colleagues found that in estrogen receptor (ER) positive breast cancer, estradiol can promote the growth of ER positive breast cancer cells by producing a large number of CXCL12 and regulating CXCR4 and CXCR7 receptors of CXCL12, and the chemokine receptor CXCR4 can enhance the response of cells to estradiol 30 .…”