Improvement of CXCR4 tracer specificity for PET imaging

Abstract

Tumors expressing the chemokine receptor CXCR4 have been reported to be more aggressive and to produce more metastatic seeding in specific organs, such as the bone marrow. However, evaluation of tumors for CXCR4 expression requires testing of ex vivo biopsy samples, and is not routinely done in cancer management. In prior work to address this issue, we and others have developed tracers for positron emission tomography (PET) that targeted CXCR4, but in addition to binding to CXCR4 these tracers also bound to red blood cells (and to other unrelated targets) in vivo. Here we report two new tracers based on the CXCR4 peptide antagonist 4F-benzoyl-TN14003 (T140) that bind to CXCR4, but not to undesired targets. These tracers, NOTA-NFB and DOTA-NFB, show slight reductions in both 1) binding affinities for CXCR4 and 2) inhibition of CXCL12 induced migration, compared to T140, in vitro. Both NOTA-NFB and DOTA-NFB specifically accumulate in CXCR4-positive, but not CXCR4-negative, tumor xenografts in mice and allow clear visualization of CXCR4 expression by PET. Evaluation of NOTA-NFB and DOTA-NFB for their potential to mobilize immune cells and progenitor cells from the bone marrow to the peripheral blood revealed slightly reduced, but still comparable, results to the parent molecule T140. The tracers reported here may allow the evaluation of CXCR4 expression in primary tumors and metastatic nodules, and enable better informed, more personalized treatment for patients with cancer.

Introduction

Chemokine receptors (CKRs) are a subfamily of seven-transmembrane domain, G-protein coupled receptors, that upon binding to their chemokine ligands, guide the migration of cells within an organism [1]. More than 40 chemokines and 19 chemokine receptors have been identified in humans [2], [3], [4], [5]. Among CKRs, CXCR4 is unusual in the breadth of its biological activities beyond leukocyte recruitment, which include roles in the development of the hematopoietic, cardiovascular, and nervous systems during embryogenesis [6]. The natural human ligand of CXCR4 is the chemokine CXCL12/SDF-1, and both ligand and receptor have been highly conserved during evolution [7], [8].

One of the physiological roles of CXCR4 is in directing cells to organ sites that express high levels of CXCL12, suggesting that this interaction plays a key role in the chemotaxis, homing, and retention of hematopoietic cells during homeostasis. Of particular interest, CXCR4–CXCL12 plays a role in stem cell homing to and retention in the bone marrow [9]. Previous studies have shown that mobilization of stem cells from the bone marrow by treatment with granulocyte colony-stimulating factor (G-CSF) involves degradation of CXCL12 in the bone marrow, promoting cell egress [10]. Moreover, specific disruption of the interaction between CXCR4 and CXCL12 in the bone marrow, by use of antagonists, acts additively with G-CSF treatment to improve stem cell mobilization [1], [11], [12], [13].

In addition to its extensive physiological roles, CXCR4 has also been found to be expressed by various human cancers including breast, prostate, lung, colon and multiple myeloma [14], [15], [16], [17], [18], [19], [20], [21], and has been suggested to be involved in the process of cancer cell metastasis [22], [23]. CXCR4 expression can be induced by a number of transcription factors, including hypoxia inducible factor-1 (HIF-1), NF-κB and oncoproteins PAX3-FKHR and RET/PTC, which have been shown to be expressed by cancers [14], [24]. There is evidence, too, that high levels of CXCR4 expression in cancers correlate with poor prognosis, and with resistance to chemotherapy. In the case of cancers of hematopoietic origin, this is thought to occur in part due to enhanced interactions between cancers and bone marrow stroma [20], [21].

Collectively, the data on CXCR4 in cancer suggest that this receptor increases tumor cell survival and/or growth and/or metastasis, making it a potentially attractive therapeutic target [14], [24], [25]. Currently, CXCR4 expression in a tumor can only be evaluated using excised tissue specimens, which limits data to the site of biopsy and to a single point in time. The development of CXCR4-specific positron emission tomography (PET) tracers would enable non-invasive evaluation of CXCR4 expression in whole tumors and, thereby, could be expected to promote personalized treatment for patients with cancer.

4-F-benzoyl-TN14003 (T140) is a high affinity CXCR4 peptide antagonist consisting of 14 amino acids, one disulfide bridge between Cys⁴ and Cys¹³, an amidated C terminus, and a 4-fluorobenzoyl group at the N-terminus (Fig. 1). Administration of T140 to mice induces in vivo mobilization of stem cells from the bone marrow, which is additive in effect with G-CSF treatment [12]. We have previously labeled T140 with ¹⁸F and found that it binds to CXCR4 in tumors, but this was masked in vivo because it also bound to red blood cells (RBCs) [26]. Moreover, the radiolabeling of T140 with ¹⁸F was laborious and time consuming, limiting its practicality in clinical settings.

To address both issues we have developed several derivatives of T140 that add a chelator to the peptide to make it amenable to labeling with ⁶⁴Cu in high radiochemical yield. Two of these derivatives (⁶⁴Cu-DOTA-NFB and ⁶⁴Cu-NOTA-NFB, Fig. 1) were found to bind specifically to CXCR4, without binding to RBC. ⁶⁴Cu-DOTA-NFB and ⁶⁴Cu-NOTA-NFB were then evaluated for their potential for in vivo imaging of CXCR4 in tumor-bearing mice. The results shown here demonstrate the usefulness of PET for evaluating a drug's binding profile in vivo and in helping to guide the elimination of “off-target” interactions during drug development.