Treatment of HER2-positive breast carcinomatous meningitis with intrathecal administration of α-particle-emitting 211At-labeled trastuzumab

doi:10.1016/j.nucmedbio.2009.04.003

Nuclear Medicine and Biology

Volume 36, Issue 6, August 2009, Pages 659-669

https://doi.org/10.1016/j.nucmedbio.2009.04.003 Get rights and content

Abstract

Introduction

Carcinomatous meningitis (CM) is a devastating disease characterized by the dissemination of malignant tumor cells into the subarachnoid space along the brain and spine. Systemic treatment with monoclonal antibody (mAb) trastuzumab can be effective against HER2-positive systemic breast carcinoma but, like other therapies, is ineffective against CM. The goal of this study was to evaluate the therapeutic effect of α-particle emitting ²¹¹At-labeled trastuzumab following intrathecal administration in a rat model of breast carcinoma CM.

Methods

Athymic rats were injected intrathecally with MCF-7/HER2-18 breast carcinoma cells through a surgically implanted indwelling intrathecal catheter. In Experiment 1, animals received 33 or 66 μCi ²¹¹At-labeled trastuzumab, cold trastuzumab or saline. In Experiment 2, animals were inoculated with a lower tumor burden and received 46 or 92 μCi ²¹¹At-labeled trastuzumab or saline. In Experiment 3, animals received 28 μCi ²¹¹At-labeled trastuzumab, 30 μCi ²¹¹At-labeled TPS3.2 control mAb or saline. Histopathological analysis of the neuroaxis was performed at the end of the study.

Results

In Experiment 1, median survival increased from 21 days for the saline and cold trastuzumab groups to 45 and 48 days for 33 and 66 μCi ²¹¹At-labeled trastuzumab, respectively. In Experiment 2, median survival increased from 23 days for saline controls to 68 and 92 days for 46 and 92 μCi ²¹¹At-labeled trastuzumab, respectively. In Experiment 3, median survival increased from 20 days to 29 and 36 days for animals treated with ²¹¹At-labeled TPS3.2 and ²¹¹At-labeled trastuzumab, respectively. Long-term survivors were observed exclusively in the ²¹¹At-trastuzumab-treated groups.

Conclusion

Intrathecal ²¹¹At-labeled trastuzumab shows promise as a treatment for patients with HER2-positive breast CM.

Introduction

Carcinomatous meningitis (CM) is a devastating disease characterized by the dissemination of malignant tumor cells within the leptomeningeal space and metastatic spread throughout the cerebrospinal fluid (CSF) compartment along the brain and spine. The disease occurs in approximately 4–15% of patients with solid tumors and is most frequently diagnosed in patients with breast carcinoma (27–50%), lung carcinoma (22–36%), lymphoma or leukemia (5–15%) as well as tumors of the central nervous system (CNS), mostly notably, medulloblastoma (30%) [1], [2]. Developing better treatments for CM accompanying breast cancer is particularly important not only because of its relative frequency but also due to the fact that its incidence appears to be increasing in part because systemic disease has become more effectively managed [3]. The prognosis for patients with CM is grim with a median survival of only 2–6 months. This is due in large part to the fact that external beam radiotherapy and chemotherapy are generally self-limited in this setting by their cytotoxicity to normal tissues, particularly those of the CNS. An attractive approach to increasing the specificity of CM treatment is targeted radiotherapy, in which a vector such as a monoclonal antibody (mAb) is utilized to selectively deliver a radionuclide to cancer cells.

With regard to the choice of vehicle for this purpose, the epidermal growth factor receptor tyrosine kinase known as both ErbB2 and HER2/neu is a potentially valuable target for cancer therapeutics. The p185 transmembrane protein HER2 oncogene product is overexpressed in about 25% of breast carcinomas and other malignancies but only at low levels on normal tissues [4], [5], [6]. Trastuzumab (Herceptin, Genentech, South San Francisco, CA, USA) is a humanized mAb that specifically binds to a cysteine-rich motif within the extracellular domain of this p185 protein [7]. Systemically administered Trastuzumab is broadly utilized, primarily in combination with chemotherapy, for the treatment of patients with HER2-positive breast carcinoma with responses observed in about half of the time [8], [9].

For unknown reasons, HER2-positive breast carcinoma patients have a relatively high incidence of CM [3]. The impermeability of the blood-brain barrier (BBB) hinders the delivery of systemically administered macromolecules to lesions located within the CNS, such as brain metastasis or CM, in a therapeutically significant manner: following intravenous administration, the CSF concentration of trastuzumab remains 300-fold lower than its systemic concentration [10]. Compartmental administration (intratumoral, intrathecal) bypasses the BBB, thereby allowing for significantly higher doses available for binding to HER2-positive tumor cells. This has lead to a number of case reports investigating the therapeutic effectiveness of high-dose intrathecal trastuzumab, with two of five patients treated surviving for more than 6 months [11], [12], [13], [14], [15].

We hypothesize that the efficacy of intrathecal trastuzumab could be enhanced by combining the mAb with a radionuclide possessing emission characteristics that are well matched to the geometrical features of CM. Because leptomeningeal spread of malignancies present as free-floating cancer cells in the CSF and sheet-like deposits on compartmental walls, radionuclides emitting short range radiation are recommended to minimize radiation dose to the spinal cord [16]. Alpha particles such as those emitted by ²¹¹At have a range in tissue of only a few cell diameters and, thus, might be ideally suited to this purpose. In addition, the 7.2-h half-life of this radiohalogen reduces the risk of systemic toxicity after CSF protein resorption into the general circulation. Finally, as a consequence of the high linear energy transfer nature of α-particles, the cytotoxicity of ²¹¹At-labeled compounds is considerably higher than those labeled with β-emitters such as ¹³¹I and ⁹⁰Y in routine use for clinical radioimmunotherapy [17].

In the present study, we describe a rat model for HER2-positive breast carcinoma CM. This model was utilized to evaluate the therapeutic potential of ²¹¹At-labeled trastuzumab and our results indicate that significant survival prolongation could be obtained after intrathecal administration of this targeted radiotherapeutic.

Section snippets

Antibodies

Trastuzumab was obtained from the Duke University Medical Center hospital pharmacy and was dialyzed overnight into 100-mM pH 8.5 borate buffer prior to labeling. The chimeric human/murine TPS3.2 mAb, produced as described previously [18], was treated in similar fashion and served as a control. Fluorescence-activated cell sorting analysis of this anti-dansyl IgG₂ mAb confirmed its lack of reactivity to the HER2-expressing cell line used in these studies (data not shown).

Labeling mAbs with ²¹¹At

Astatine-211 was produced

Breast carcinomatous meningitis model

The effect of altering the number and volume of inoculated cells was evaluated in preliminary experiments where it was found that CM could be established reproducibly with volumes of 80–100 μl and 0.5–1.25×10⁷ MCF-7/HER2-18 cells. As observed previously, in order to use this cell line to establish intracerebral xenografts in this rat strain [22], consistent tumor growth required use of a continuous-release 17β-estradiol pellet. At these cell doses, animals developed progressive neurological

Discussion

Better treatments for malignancies that metastasize to the subarachnoid space are critically needed because current therapies are largely ineffective [1], [2]. Although radiation has been utilized in patients with CM, external beam therapy can rarely achieve a meaningful therapeutic effect because of dose-limiting toxicity to the normal neuraxis [3]. Targeted radiotherapy is attractive in this setting because of the potential for achieving high levels of tumor cell irradiation while sparing

Acknowledgments

This work was supported by grants CA42324 and NS20023 from the National Institutes of Health and Grant DOE-FG02-08ER64697 from the Department of Energy. Dr. Abraham Boskovitz was supported by the SICPA Foundation (Prilly, Switzerland), the Swiss Cancer League and the 450^th Anniversary of the University of Lausanne Foundation (Lausanne, Switzerland). We thank Dr. Xiao-Guang Zhao for his assistance in preparing the ²¹¹At-labeled mAbs.

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Akabani et al. [204] pointed out that the relative biological effect of 211At-trastuzumab was twice higher than that of external irradiation therapy, and it was able to inhibit the proliferation of HER2-positive tumor cells. Furthermore, the therapeutic effect of 211At-trastuzumab on carcinomatous meningitis was verified in murine models [205]. It had been proved that the median survival of tumor-bearing mice increased from 21 days in saline group to 45 and 48 days after intracranial administration of 1.22 and 2.44 MBq of 211At-trastuzumab, respectively.
Astatine-211 (²¹¹At, t_1/2 = 7.21 h) emitting two α particles with energies of 5.87 and 7.45 MeV, can lead to a high linear energy transfer (LET = 98.84 keV/µm) and short tissue range (50∼90 µm). Since the 1950s, ²¹¹At had stepped into endoradiotherapy and has always been regarded as one of the most promising α-emitters for targeted-alpha therapy (TAT) in various malignancies. In the past two decades, ²¹¹At related radiopharmaceuticals have achieved great progress in the studies of basic physicochemical properties of astatine, ²¹¹At labeling strategies, preclinical and clinical studies, producing profound effects in nuclear medicine. This work will give a panorama of ²¹¹At-related researches in the recent 20 years, which will cover both the fundamental insights of ²¹¹At radiochemistry and applied labeling compounds. It can provide some important hints for the studies of TAT and other radiopharmaceuticals applied in tumor radiotherapy.
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The tumor microenvironment (TME) is a dynamic structure that is highly interactive with cancer cells and is shaped to a large extent by these cells. Abnormal vasculature, hypoxia, acidic extracellular pH, an altered extracellular matrix, and stromal cells such as cancer-associated fibroblasts, macrophages, and immune cells are some of the characteristics of the TME. These characteristics contribute significantly to cancer progression but also provide novel opportunities for cancer-specific therapeutic strategies. The fluidity of the TME, with its spatial and temporal heterogeneities, makes noninvasive molecular and functional imaging technologies highly important to image TME components and to develop image-guided theranostic strategies for cancer-specific precision treatments. Here we have reviewed the characteristics of the TME, the imaging modalities available to visualize the TME components, and the theranostic strategies that are available for image-guided therapy of the TME.
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An advantage of 211At is the ability to label a broad range of molecules for uses as therapeutic radiopharmaceuticals, such as 211At-labeled macromolecules.12 A preclinical study of [211At]trastuzumab targeted to HER2-expressing leptomeningeal breast cancer shows promising results.20 Similarly, 211At-labeled monoclonal antibodies can be targeted to leukemia (CD-45), lymphoma (CD-20), and multiple myeloma (CD38), with active clinical trials in leukemia and promising pre-clinical studies in lymphoma and multiple myeloma.21-24
Radiation oncologists and nuclear medicine physicians have seen a resurgence in the clinical use of radiopharmaceuticals for the curative or palliative treatment of cancer. To enable the discovery and the development of new targeted radiopharmaceutical treatments, the United States National Cancer Institute has adapted its clinical trial enterprise to accommodate the requirements of a development program with investigational agents that have a radioactive isotope as part of the studied drug product. One change in perspective has been the consideration of investigational radiopharmaceuticals as drugs, with maximum tolerable doses determined by normal organ toxicity frequency like in drug clinical trials. Other changes include new clinical trial enterprise elements for biospecimen handling, adverse event reporting, regulatory conduct, writing services, drug master files, and reporting of patient outcomes. Arising from this enterprise, the study and clinical use of alpha-particle and beta-particle emitters have emerged as an important approach to cancer treatment. Resources allocated to this enterprise have brought forward biomarkers of molecular pathophysiology now used to select treatment or to evaluate clinical performance of radiopharmaceuticals. The clinical use of diagnostic and therapeutic radionuclide pairs is anticipated to accelerate radiopharmaceutical clinical development.
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Indeed, cerebrospinal fluid (CSF) levels of trastuzumab in patients given i.v. infusions were 49–420 times lower than matched serum levels [4]. Trastuzumab IT administration in rat models of CNS metastatic HER2-BC was safe without toxicity events and was superior to systemic delivery or to isotype-matched control antibody in terms of survival [5–7]. In a recent review of the literature pooling 17 case reports of IT administration of trastuzumab for MC in HER2-BC, Zagouri et al. reported no serious adverse events in 88.2% of cases and found significant clinical improvement in 68.8% of cases [8].
Leptomeningeal carcinomatosis (MC) is commonly associated with HER2-positive breast cancer (HER2-BC), with a poor prognosis and no standardised treatment. We conducted a phase I dose-escalation study of intrathecal (IT) administration of trastuzumab in HER2-BC patients with MC to determine the maximum tolerated dose (MTD), which was based on both the achievement of a trastuzumab intra-cerebrospinal fluid concentration close to a conventional therapeutic plasma concentration (30 mg/L) and/or dose-limiting toxicity (DLT).
The protocol planned IT administration of trastuzumab (30 mg, 60 mg, 100 mg or 150 mg dose levels) once a week, over the course of at least 4 weeks. Sixteen patients with MC from HER2-BC received IT trastuzumab. Intra-cerebrospinal fluid samples were obtained before each injection for pharmacokinetics.
We did not observe DLT of IT trastuzumab. Eleven patients had no toxicity attributed to IT trastuzumab. For 60 mg or higher dose levels, minor toxicities attributed to IT trastuzumab included headache (2 patients), nausea (2 patients), vomiting (1 patient), cervical pain (1 patient) and peripheral neuropathy (1 patient). Two patients experienced immediate toxicity including headache or vomiting. The mean residual intra-cerebrospinal fluid concentration of trastuzumab was 27.9 mg/L for the 150 mg dose level. Three patients achieved a clinical response, seven patients had stable disease and four patients had progressive disease.
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The relative biological effectiveness of 211At-labeled trastuzumab was about 10 times higher than that of conventional external beam therapy, with significant reduction in survival achieved with only a few 211At atoms bound per cell. A subsequent study was performed in a HER2-positive breast carcinomatous meningitis model to evaluate the therapeutic efficacy of a single intrathecal injection of 211At-labeled trastuzumab [15]. Significant prolongation in median survival with some long-term survivors was observed; however, even with direct injection into the intrathecal compartment, histopathological analyses revealed that regions of the neuroaxis had escaped treatment in some animals.
Derived from heavy chain only camelid antibodies, ~ 15-kDa single-domain antibody fragments (sdAbs) are an attractive platform for developing molecularly specific imaging probes and targeted radiotherapeutics. The rapid tumor accumulation and normal tissue clearance of sdAbs might be ideal for use with ²¹¹At, a 7.2-h half-life α-emitter, if appropriate labeling chemistry can be devised to trap ²¹¹At in cancer cells after sdAb binding. This study evaluated two reagents, [²¹¹At]SAGMB and iso-[²¹¹At]SAGMB, for this purpose.
[²¹¹At]SAGMB and iso-[²¹¹At]SAGMB, and their radioiodinated analogues [¹³¹I]SGMIB and iso-[¹³¹I]SGMIB, were synthesized by halodestannylation and reacted with the anti-HER2 sdAb 5F7. Radiochemical purity, immunoreactivity and binding affinity were determined. Paired-label internalization assays on HER2-expressing BT474M1 breast carcinoma cells directly compared [¹³¹I]SGMIB-5F7/[²¹¹At]SAGMB-5F7 and iso-[¹³¹I]SGMIB-5F7/iso-[²¹¹At]SAGMB-5F7 tandems. The biodistribution of the two tandems was evaluated in SCID mice with subcutaneous BT474M1 xenografts.
Radiochemical yields for Boc₂-iso-[²¹¹At]SAGMB and Boc₂-[²¹¹At]SAGMB synthesis, and efficiencies for coupling of iso-[²¹¹At]SAGMB and [²¹¹At]SAGMB to 5F7 were similar, with radiochemical purities of [²¹¹At]SAGMB-5F7 and iso-[²¹¹At]SAGMB-5F7 > 98%. iso-[²¹¹At]SAGMB-5F7 and [²¹¹At]SAGMB-5F7 had immunoreactive fractions > 80% and HER2 binding affinities of less than 5 nM. Internalization assays demonstrated high intracellular trapping of radioactivity, with little difference observed between corresponding ²¹¹At- and ¹³¹I-labeled 5F7 conjugates. Higher BT474M1 intracellular retention was observed from 1-6 h for the iso-conjugates (iso-[²¹¹At]SAGMB-5F7, 74.3 ± 2.8%, vs. [²¹¹At]SAGMB-5F7, 63.7 ± 0.4% at 2 h) with the opposite behavior observed at 24 h. Peak tumor uptake for iso-[²¹¹At]SAGMB-5F7 was 23.4 ± 2.2% ID/g at 4 h, slightly lower than its radioiodinated counterpart, but significantly higher than observed with [²¹¹At]SAGMB-5F7. Except in kidneys and lungs, tumor-to-normal organ ratios for iso-[²¹¹At]SAGMB-5F7 were greater than 10:1 by 2 h, and significantly higher than those for [²¹¹At]SAGMB-5F7.
These ²¹¹At-labeled sdAb conjugates, particularly iso-[²¹¹At]SAGMB-5F7, warrant further evaluation for targeted α-particle radiotherapy of HER2-expressing cancers.

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^☆: This work was supported by Grants CA42324 and NS20023 from the National Institutes of Health and Grant DOE-FG02-08ER64697 from the Department of Energy.

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Treatment of HER2-positive breast carcinomatous meningitis with intrathecal administration of α-particle-emitting 211At-labeled trastuzumab☆

Abstract

Introduction

Methods

Results

Conclusion

Introduction

Section snippets

Antibodies

Labeling mAbs with 211At

Breast carcinomatous meningitis model

Discussion

Acknowledgments

Semin Oncol

Semin Oncol

Lancet Oncol

Clin Breast Cancer

Annals Oncol

Nucl Med Biol

Nucl Med Biol

J Neurosci Methods

Int J Radiat Oncol Biol Phys

Nucl Med Biol

Primary neoplasms of the central nervous system in adults

Neoplastic meningitis

J Clin Oncol

Expression of the HER-2/neu proto-oncogene in normal human adult and fetal tissues

Oncogene

Overexpression of HER-2/neu is associated with poor survival in advanced epithelial ovarian cancer

Cancer Res

Expression of the p185 encoded by HER2 oncogene in normal and transformed human tissues

Int J Cancer

Nonclinical studies addressing the mechanism of action of trastuzumab (Herceptin)

Semin Oncol

Clinical trials of Herceptin (trastuzumab)

Eur J Cancer

Trastuzumab in CSF

J Clin Oncol

Treatment of HER2-positive breast carcinomatous meningitis with intrathecal administration of α-particle-emitting ²¹¹At-labeled trastuzumab☆

Labeling mAbs with ²¹¹At