Radiolabeling of trastuzumab with 177Lu via DOTA, a new radiopharmaceutical for radioimmunotherapy of breast cancer

doi:10.1016/j.nucmedbio.2009.01.015

Nuclear Medicine and Biology

Volume 36, Issue 4, May 2009, Pages 363-369

https://doi.org/10.1016/j.nucmedbio.2009.01.015 Get rights and content

Abstract

Aim

Trastuzumab is a monoclonal antibody that is used in treating breast cancer. We labeled this monoclonal antibody with lutetium-177 and performed in vitro quality control tests as a first step in the production of a new radiopharmaceutical.

Material and Methods

Trastuzumab was labeled with lutetium-177 using DOTA as chelator. Radiochemical purity and stability in buffer and human blood serum were determined using thin layer chromatography. Immunoreactivity and toxicity of the complex were tested on MCF7 breast cancer cell line.

Results

The radiochemical purity of the complex was 96±0.9%. The stabilities in phosphate buffer and in human blood serum at 96 h postpreparation were 93±1.2% and 85±3.5%, respectively. The immunoreactivity of the complex was 89±1.4%. At a concentration of 1 nM, the complex killed 70±3% of MCF7 cells. At 1.9 nM, 90±5% of the cells were killed.

Conclusions

The results showed that the new complex could be considered for further evaluation in animals and possibly in humans as a new radiopharmaceutical for use in radioimmunotherapy against breast cancer.

Introduction

Cancer treatment using radioimmunotherapy has been the focus of much research in the last two decades. In radioimmunotherapy, a radioisotope is coupled to a monoclonal antibody to form a tumor-specific target agent. Trastuzumab, commonly known as Herceptin, has been used in the treatment of breast cancer as a US Food and Drug Administration-approved drug since 1998 [1]. It is a humanized IgG1 monoclonal antibody directed against the extracellular domain of the human epidermal growth factor receptor 2 (HER2) and is thus used in patients whose tumors overexpress this receptor. Amplification of this receptor occurs in 20–30% of early-stage breast cancers [2], [3], [4]. Trastuzumab works by attaching itself to the HER2 protein and preventing the epidermal growth factor from reaching the breast cancer cells. This stops their cell division and attracts immune cells to destroy the target cells [5]. Trastuzumab has recently been used in radioimmunotherapy as a tumor-specific carrier [6], [7], [8]. Apparently, the efficiency of the trastuzumab–radioisotope complex is very much dependent on the type and energy of radiation emitted from the radioisotope.

Lanthanide radioisotopes are β⁻ emitters that have been demonstrated to be suitable for radioimmunotherapy of tumors [9]. Radiolanthanides are easily produced with a large-scale, low-price, and high specific activity. ¹⁷⁷Lu is a radiolanthanide with a β⁻ emission similar to ¹³¹I but with two advantages over it. The main γ-photons of ¹⁷⁷Lu (208 keV, 11% abundance) are more suitable for imaging with a gamma camera than those of ¹³¹I (364 keV, 82% abundance), and ¹⁷⁷Lu does not need special design of residualizing for labeling with internalizing monoclonal antibodies. The half-life of ¹⁷⁷Lu is 6.65 days. Due to its physical characteristics, ¹⁷⁷Lu is being increasingly used in therapeutic research studies [10], [11].

In the present study, we conjugated trastuzumab with ¹⁷⁷Lu and performed quality control tests and in vitro studies to evaluate the complex as a radiopharmaceutical for the radioimmunotherapy of human breast cancer.

Section snippets

Materials

Trastuzumab was purchased in 150-ml vials (Genentech, South San Francisco, CA, USA). ¹⁷⁷Lu was produced by bombarding ¹⁷⁶Lu₂O₃ (CAMPRO Scientific, Germany, 74%) dissolved in 1 M HCl in the Tehran Research Reactor (at 2.6×10¹³ n Cm⁻² S⁻¹ flux for 14 days).

The MCF7 breast cancer cell line (HER2 positive) was grown in RPMI 1640 supplemented with 10% FCS, 40 μg ml⁻¹ gentamycine, and 2 mM glutamine. The chemicals were purchased from Fluka Chemical Corp.

Preparation of NHS-DOTA

We used the method explained in the

The average number of chelates per antibody

On the average, 2.1–2.7 DOTA analogues were conjugated to trastuzumab and the conjugation reactions were performed at 20:1 for NHS-DOTA to trastuzumab.

Radiochemical purity

The relative counts recorded from the different segments of the chromatographic papers against the distance from the bottom of the papers are shown in Fig. 1. The highest activity is seen in the first segments showing very good radiochemical purity. As can be seen, the radiochemical purity of the complex was 96±0.9% at 2 h and pH of 7.

The stability in buffer and human blood serum

The

Discussion

Breast cancer patients are usually tested for the level of HER2 in their tumors. In normal cells, HER2 controls aspects of cell growth and division. When activated in cancer cells, HER2 accelerates tumor formation. In about 20–30% of breast cancers HER2 is overexpressed. These patients may be candidates for trastuzumab treatment in the postsurgical or metastatic setting [15].

Trastuzumab is a humanized monoclonal antibody which binds to the extracellular segment of the HER2 receptor. Cells

Conclusions

From these results, we can conclude that ¹⁷⁷Lu–DOTA–trastuzumab may be considered for further evaluation in animals and possibly in humans as a new radiopharmaceutical for use in radioimmunotherapy against breast cancer. The combination of trastuzumab and ¹⁷⁷Lu has some potential advantages over trastuzumab alone including enhanced efficacy, reduced side effects and lower cost.

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Radiolabeling of trastuzumab with 177Lu via DOTA, a new radiopharmaceutical for radioimmunotherapy of breast cancer

Abstract

Aim

Material and Methods

Results

Conclusions

Introduction

Section snippets

Materials

Preparation of NHS-DOTA

The average number of chelates per antibody

Radiochemical purity

The stability in buffer and human blood serum

Discussion

Conclusions

Nucl Med Biol

Nucl Med Biol

Nucl Med Biol

J Immunol Methods

J Immunol Methods

Semi Oncol

HER-2-Positive metastatic breast cancer: trastuzumab and beyond

Expert Opin Pharmacother

Molecular targets for breast cancer therapy and prevention

Nat Med

Overview of tyrosine kinase inhibitors in clinical breast cancer

Endocr Relat Cancer

HER-2-Targeted therapy — lessons learned and future directions

Clin Cancer Res

Radiolabeling of trastuzumab with ¹⁷⁷Lu via DOTA, a new radiopharmaceutical for radioimmunotherapy of breast cancer