Comparison of two cross-bridged macrocyclic chelators for the evaluation of 64Cu-labeled-LLP2A, a peptidomimetic ligand targeting VLA-4-positive tumors
Introduction
Membrane receptors of the integrin family are well-characterized targets in cancer cells. As a family of adhesive proteins, integrins have been reported to mediate interactions between adhesion molecules on adjacent cells and/or the extracellular matrix (ECM) and are involved in biological processes including cell migration and metastasis [1], [2]. Integrin α4β1 (also called very late antigen-4 or VLA-4) is a heterodimeric transmembrane receptor that is widely expressed on a variety of tumors. There is increasing evidence that α4β1integrin plays an important role in tumor growth, angiogenesis and metastasis [3], [4], [5], therefore, α4β1 integrin is a promising target for imaging and therapy of lymphoid malignancy cancers.
Radiolabeled receptor-targeted peptides have attracted attention for tumor diagnosis and therapy. This is primarily due to the ease of peptide synthesis, their favorable pharmacokinetic characteristics together with their flexibility in chemical modification and radiolabeling [6], [7]. LLP2A, a peptidomimetic ligand that has high affinity for α4β1 integrin, was first reported by Peng et al. as identified from a highly focused one-bead-one-compound combinatorial library [8]. They demonstrated that LLP2A specifically targeted α4β1-positive tumors in nude mouse xenografts with in vivo near infrared optical imaging.
LLP2A was also conjugated to 11-bis(carboxymethyl)-1,4,8,11-tetraaza-bicyclo[6.6.2]hexadecane (CB-TE2A), a highly stable 64Cu chelator, as 64Cu-CB-TE2A-LLP2A. In the Raji tumor-bearing mouse model, small-animal PET imaging demonstrated that 64Cu-CB-TE2A-LLP2A accumulated in tumor and spleen, which are α4β1 integrin-rich tissues [9]. In another study, Shokeen et al. showed that 64Cu-CB-TE2A-LLP2A can image α4β1 positive bone marrow-derived cells in sites of bone metastasis in a mouse model of metastatic breast cancer [10].
Although 64Cu-CB-TE2A bioconjugates show significant improvement over those with non-bridged macrocycles with respect to in vivo stability [11], one of the disadvantages of CB-TE2A is the requirement of harsh conditions for radiolabeling with 64Cu, typically 95 °C for 1–1.5 h [12]. To broaden the applications of the cross-bridged bifunctional chelator, a second generation chelator was developed by incorporating methane phosphonic acid pendant arms [13]. The substitution of methane phosphonic acid pendant arms brings higher selectivity for Cu(II) and a greater ease of 64Cu incorporation, while retaining high kinetic stability. A mixed armed chelator, CB-TE1A1P (1,4,8,11-tetraazacyclotetradecane-1-(methane phosphonic acid)-8-(methane carboxylic acid) was developed, featuring a methane phosphonic acid as well as a carboxymethyl pendant arm for conjugation to primary amines on peptides and proteins [14]. Radiolabeling of CB-TE1A1P showed that the 64Cu complex can be formed at room temperature after 30 min, achieving a radiochemical yield of > 95%. Here, we report radiochemistry as well as in vitro and in vivo characterization of 64Cu-CB-TE1A1P-LLP2A and 64Cu-CB-TE2A-LLP2A in the α4β1-positive B16F10 tumor model.
Section snippets
Materials and methods
Copper-64 (t1/2 = 12.7 hours, β+; 17.8%, Eβ + max = 656 KeV, β-, 38.4%, Eβ-max = 573 KeV) was obtained from Washington University (St. Louis, MO) and University of Wisconsin (Madison, WI). All chemicals were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO), unless otherwise specified. Aqueous solutions were prepared using ultrapure water (resistivity, 18 MΩ). Rink amide 4-methylbenzhydrylamine resin (loading, 0.77 mmol/g) and all Fmoc protected amino acid were purchased from Chem-Impex
Radiochemistry
Radiochemical purity of 96 ± 2% was achieved after 60 min at room temperature for 64Cu-CB-TE1A1P-LLP2A with specific activity of 1 mCi/μg. At 40 °C, the reaction time was significantly reduced to 15 min, while at 90 °C, complete radiolabeling was achieved in 5 min (Fig. 2). Under similar specific activity, radiochemical purity of 64Cu-CB-TE2A-LLP2A was 68% ± 3% after incubated at 90oC for 60 min. No radiolabeling for CB-TE2A-LLP2A was observed under 40 °C after 60 min.
Cell binding assays
Prior to the assays with the
Discussion
Integrin α4β1 plays an important role in the regulation of immune cell recruitment to inflamed endothelium and sites of inflammation [16]. It is a promising diagnostic target found in leukemias, lymphomas, melanomas and sarcomas [3], [17], [18]. It has been reported that malignant melanomas demonstrated an increase in the expression of α4β1 compared with benign melanocytic lesions [19]. Integrin α4β1 also participates in cell-lymphocyte interactions, retention and migration of immature
Conclusion
The bifunctional chelator CB-TE1A1P was successfully conjugated with LLP2A, a high-affinity peptidomimetic ligand against α4β1 integrin. In vivo evaluation shows that 64Cu-CB-TE1A1P-LLP2A had dramatically improved tumor-to-background contrast compared with 64Cu-CB-TE2A-LLP2A. 64Cu-CB-TE1A1P-LLP2A will serve as an excellent PET radiopharmaceutical for the imaging of α4β1 positive tumors, which include lymphoma, melanoma and multiple myeloma, and this agent has potential for improved imaging of
Acknowledgments
The authors gratefully acknowledge Chris Sherman, Jalpa Modi, Michael Zahner, Margaret Morris and Nicole Fettig for technical support and Julie Schwarz, MD, PhD for helpful discussions. This research was supported by NIH/NCI 5 R01 CA 093375 (CJA) and DOE DE-FG02-08ER64671 (Integrated Research Training Program of Excellence in Radiochemistry awarded to Michael J. Welch for partial support to MJ).
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Macrocyclic Bifunctional Chelators and Conjugation Strategies for Copper-64 Radiopharmaceuticals
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2014, Advanced Drug Delivery ReviewsCitation Excerpt :This study provided important information for non-invasive imaging of potential cancer metastasis origin. To boost the VLA-4 targeting efficacy of LLP2A and optimize its in vivo kinetics, a cross-bridged macrocyclic compound with a methane phosphonic acid pendant arm (named CB-TE1A1P) was conjugated to LLP2A [86]. CB-TE1A1P-LLP2A can react with 64Cu under mild conditions to produce stable radiolabeled compound with high specific activity, while high temperature is required for efficient radiolabeling using CB-TE2A.