¹⁷⁷Lu-Labeled humanized monoclonal antibody against human epidermal growth factor receptor 2

[[¹⁷⁷Lu]Pertuzumab]

Background

[PubMed]

The human epidermal growth factor (EGF) receptor 2 (HER2 or Erb2/neu) is known to be overexpressed in cells of certain cancers and to play a key role in promoting the growth and proliferation of primary and metastasized cancer cells, particularly in breast, ovarian, and urinary bladder cancers (1-4). HER2 is a glycoprotein that belongs to the EGF receptor (EGFR or ErbB1) family (for details regarding other HER or ErbB receptors and their ligands, see Friedlander et al. and Adams et al. (5, 6)). The ErbB receptors have four domains (designated I to IV): an extracellular region, a membrane-spanning region, an intracellular tyrosine kinase (TK), and a noncatalytic domain (7). Under normal conditions the EGFR and ErbB exist as monomers, but the binding of a ligand to the extracellular domain of the receptor results in conformational changes that promote the interaction of receptor domains I and III (7, 8). This interaction between two receptor domains leads to the formation of homodimers or heterodimers of the receptor and activation of the intracellular tyrosine kinase (TK). Subsequently, a complex intracellular signaling pathway may be activated, which results in malignant transformation of cells, cancer progression and survival, possible resistance to anticancer drugs, and the formation of tumors (6). In addition, overexpression or mutations of the ErbB1 or ErbB2 receptors have also been shown to promote oncogenic transformations of cells (5).

Among the ErbB receptors, ErbB2 has been shown to be the preferred receptor for the formation of heterodimers with other ErbBs (ErbB1, ErbB3, and ErbB4) because it has an open extracellular domain confirmation that facilitates the formation of potent, mitogenic heterodimers (5). Formation of the ErbB2/ErbB3 heterodimers has been reported in breast, prostate, lung, and colorectal cancers (5, 9-12). Because ErbB receptors have been shown to have a prominent role in the development of cancers, these receptors have been targeted with different antibodies (Abs) for the molecular therapy of this disease (5). Among the various Abs directed against the EGF/ErbB receptor, trastuzumab and pertuzumab specifically bind to and inhibit ErbB2 receptor activity (5).

Trastuzumab is a humanized monoclonal antibody (MAb) that inhibits cell proliferation by binding to the extracellular domain of the ErbB2 receptor and by blocking activation of the intracellular TK; however, the exact mode by which it inhibits the TK is unknown, and several mechanisms have been proposed for its activity (5, 13). This MAb is commercially available in the United States and has been approved by the United States Food and Drug Administration for the treatment of breast cancer; some patients undergoing treatment with this MAb develop resistance against it or may experience cardiac toxicity (5). Pertuzumab is a new class of humanized MAbs against ErbB2 and is known as a receptor dimerization inhibitor. On binding to the dimerization domain of the ErbB2 receptor, pertuzumab sterically hinders receptor dimerization and inhibits the signal transduction pathway (7). It has been tested in vitro and in preclinical animal studies, and it is being evaluated in several clinical trials to treat a variety of cancers as a monotherapy or in combination with other drugs and MAbs (14-19). Pertuzumab has been labeled with radioactive lutetium (¹⁷⁷Lu) to obtain [¹⁷⁷Lu]pertuzumab and used for biodistribution and imaging studies in mice (20, 21).

Synthesis

[PubMed]

The synthesis, development, purification, and characterization of pertuzumab have been described in detail by Adams et al. (6).

Pertuzumab was radiolabeled as described by Persson et al. (20). Briefly, the MAb was conjugated to 2-(p-isothiocyanatobenzyl)-cyclohexyl-diethylenetriamine pentaacetic acid (CHX-A DTPA) in 0.7 M borate buffer (pH 9.2) overnight at 37°C. The conjugated MAb was purified on a NAP-5 size exclusion column in sodium acetate buffer (pH 5.5) and divided into ten batches for storage (the storage conditions were not described) until required for chelation with ¹⁷⁷Lu. Chelation was performed for 30 min at room temperature, and purity of the chelated product was checked with instant thin-layer chromatography (ITLC). Chelation efficiency of the reaction was reported to be ~90%, and the product purity was ~99%. The Rf values for the various products of the chelation reaction on ITLC analysis were not provided in the publication. The specific activity of [¹⁷⁷Lu]pertuzumab used for the in vitro studies was reported to be 1 MBq/pmol Ab (0.027 mCi/pmol Ab). For imaging studies the specific activity of [¹⁷⁷Lu]pertuzumab was 45 MBq/pmol Ab (1.21 mCi/pmol Ab). The stability of ¹⁷⁷Lu chelated to pertuzumab was reported to be 96 ± 5% when stored overnight in presence of excess ethylenediaminetetraactetate. When stored in phosphate-buffered saline the stability of chelated ¹⁷⁷Lu was 93 ± 4% at 37°C and 100% at 4°C.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

The dissociation constant (K_d) for native pertuzumab and CHX-conjugated pertuzumab was determined with Biacore analysis using a CM5 Sensor Chip with the immobilized HER2 receptor (20). The native pertuzumab had a K_d of 2.2±0.4 nM, and the CHX-conjugated MAb had a K_d of 1.8 ± 1.1 nM. The affinity constant was determined to be 4.1 ± 0.7 nM using a saturation experiment with SKOV-3 cells exposed to [¹⁷⁷Lu]pertuzumab. The [¹⁷⁷Lu]pertuzumab retention in the SKOV-3 cells was 90 ± 2% after 20 h and dropped to 51 ± 3% at 90 h. Native pertuzumab was reported to block the uptake of [¹⁷⁷Lu]pertuzumab by the SKOV-3 cells, indicating that the labeled MAb was specific for HER2 (20).

Animal Studies

Human Studies

[PubMed]

No publications are currently available.

Supplemental Information

[Disclaimers]