In vitro and in vivo characterization of 177Lu-huA33: A radioimmunoconjugate against colorectal cancer
Introduction
Colorectal cancer is the second most common form of cancer in Europe [1] and the third most common, in terms of incidence, worldwide [2]. This type of cancer accounts for 10–12% of cancer deaths in Europe [3] and in the United States [4]. While surgery can be curative (with or without adjuvant chemotherapy [5], [6]), the disease frequently presents in an advanced stage and recurrent disease often develops, frequently in the form of distant metastases, most often in liver and lung [7]. The liver is the most frequent site of failure, with up to 50% of colorectal cancer patients developing hepatic metastases [8], [9]. Liver resection is the main therapeutic option for hepatic metastases, and advances in the field of surgery have led to an increasing amount of patients being eligible for tumor resection [10]. As a complement to surgery, radiofrequency ablation [11] or chemotherapy may be used to increase the survival rate [10]. It is also possible to perform repeated liver resections in the case of further hepatic recurrence [9], but a large number of patients still do not survive beyond 5 years [1], [10]. Thus, further development of new therapeutic strategies is needed for more effective treatment of metastatic disease.
Targeted radiotherapy is an appealing concept with the potential to improve cancer therapy. A targeting agent delivers a therapeutic radionuclide specifically to tumor cells by binding to molecular structures present, in the ideal situation, on tumor cells only. In this way, normal healthy tissue will be spared. Radioimmunotherapy (RIT), where antibodies are used as targeting agents, is a promising field of study in this area. RIT has been successful for treatment of hematological cancers, for example, lymphoma, but has shown less favorable results in patients with solid tumors, such as colorectal cancer [12]. For therapy of colorectal cancer, RIT might, therefore, probably best be utilized in an adjuvant setting for eradication of small metastases, as has previously been indicated in clinical studies using an anti-CEA antibody [13], [14].
The humanized monoclonal antibody A33 (huA33), directed against the antigen with the same name, is a promising targeting agent for RIT of metastatic colorectal carcinoma. The A33 antigen is a transmembrane glycoprotein [15] of the junctional adhesion molecule family [16], [17]. It is homogenously expressed in more than 95% of all colorectal carcinomas, with the same level of expression in metastases [18], which is an important quality for an RIT target. The expression of the antigen in normal tissue is very limited; almost the only organ expressing the A33 antigen is normal colonic mucosa [18].
After binding to tumor cells, huA33 shows excellent retention in tumor tissue. In clinical studies with radiolabeled murine A33, the radioactive label was still traceable in tumor tissue up to 6 weeks after injection of the antibody, while it was cleared from normal tissue within 1 week [19]. The same fast clearance from normal colon has been stated for huA33, with a clearance rate that corresponds to the turnover time of normal colonocytes [20].
We have, in earlier studies, labeled huA33 with the α-emitter 211At and investigated the in vitro [21] and in vivo (manuscript in preparation) characteristics of the 211At-huA33 conjugate. Due to the short half-life of 211At (T1/2=7.2 h), it might, however, not be the ideal therapeutic nuclide for use together with huA33. As mentioned above, huA33 displays very good retention in tumor tissue, but it is released more rapidly from normal cells. To be able to take full advantage of this antibody characteristic for therapeutic purposes, one would need a radionuclide with a sufficiently long half-life in order to maximize the tumor-to-normal tissue dose ratio.
The β-emitter 177Lu (maximum energy, 497 keV) is a promising therapeutic radionuclide with a half-life of 6.7 days that should suit the good tumor retention of huA33. As a low-energy β-emitter, it should be well suited for treatment of subclinical metastases. Some of the advantages of 177Lu are that it is commercially available and that its γ-emission, while suitable for imaging [22], contributes to less than 10% of total body irradiation due to low abundance.
The purposes of this study were to label huA33 with 177Lu via the chelator CHX-Aʺ-DTPA and to determine the in vitro and in vivo characteristics of the 177Lu-CHX-Aʺ-huA33 (177Lu-huA33) conjugate.
Section snippets
Materials
The humanized IgG1 antibody A33 (kindly provided by the Ludwig Institute for Cancer Research, New York, USA) was used in all experiments. The chelator CHX-Aʺ-DTPA from Macrocyclics (Dallas, USA) was used in the labeling reactions. Lutetium-177 (3.03 GBq/ml) was purchased from I.D.B. Holland B.V. (The Netherlands). Change of buffer during the radiolabeling process was made using NAP-5 columns (cutoff, 5 kDa; Amersham Biosciences AB, Uppsala, Sweden). Radioactivity measurements were made in a
In vitro experiments
huA33 was successfully labeled with 177Lu, using the chelator CHX-Aʺ-DTPA. The specific binding of 177Lu-huA33 to SW1222 cells is shown in Fig. 1. The 177Lu-huA33 conjugate was found to bind specifically to SW1222 cells since 177Lu-huA33 could be displaced by adding an excess amount of unlabeled huA33.
Fig. 2 shows the continuous uptake of 177Lu-huA33 in SW1222 cells. The uptake increases continuously during the time of the study, reaching approximately 35% of added activity after 72 h of
Discussion
RIT has been successfully used against CD-20 lymphoma [23]. While RIT of solid tumors has been much further behind in development, this may well be a function of study design—most patients studied had advanced, bulky disease [24]. Goldenberg [12] has demonstrated the potential for RIT in the minimal residual disease situation, and RIT will hopefully become an alternative for adjuvant therapy of colorectal carcinoma. As a targeting agent for RIT of colorectal carcinoma, the huA33 is a promising
Acknowledgments
The authors would like to thank the Ludwig Institute for Cancer Research, New York, NY, USA, for its kind donation of huA33 and SW1222 cells. We would also like to thank Veronika Eriksson for valuable laboratory assistance.
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