APhase I Trial of HumanizedMonoclonal AntibodyA 33 in Patients with Colorectal Carcinoma : Biodistribution , Pharmacokinetics , and QuantitativeTumor Uptake

Purpose:To determine the in vivo characteristics of huA33, a CDR-grafted humanized antibody against theA33antigen,wehave conductedanopen-label, dose escalation, biopsy-basedphase I trial of huA33 in patients with colorectal carcinoma. Experimental Design:Patients with colorectal carcinoma were infused with [I]huA33 (400 MBq: 10 mCi) and [I]huA33 (40 MBq: 1mCi) 1week before surgery.There were four huA33 dose levels (0.25, 1.0, 5.0, and 10 mg/m). Adverse events, pharmacokinetics, biodistribution, tumor biopsies, and immune responses to huA33 were evaluated. Results:There were 12 patients entered into the trial (6 males and 6 females; age range, 39-66 years). No dose-limiting toxicity was observed. The biodistribution of huA33 showed excellent uptake of [I]huA33 in metastatic colorectal carcinoma. Pharmacokinetic analysis showed no significant difference in terminal half-life (T1/2h) between dose levels (mean F SD, 86.92 F 22.12 hours). Modeling of colon uptake of huA33 showed aT1/2 of elimination of 32.4 F 8.1 hours. Quantitative tumor uptake ranged from 2.1 10 3 to 11.1 10 3 %ID/g, and tumor/normal tissue and tumor/serum ratios reached as high as 16.3:1 and 4.5:1, respectively. Biosensor analysis detected low-level human anti-human antibody responses in four patients following huA33 infusion. Conclusions: huA33 shows selective and rapid localization to colorectal carcinoma in vivo and penetrates to the center of large necrotic tumors, and colon elimination half-life of huA33 is equivalent to basal colonocyte turnover.The excellent targeting characteristics of this humanized antibody indicate potential for the targeted therapy of metastatic colorectal cancer in future trials. Colorectal cancer is one of the most common cancers in the western world and is the second most common cause of cancer-related mortality (1–3). Adjuvant chemotherapy, radiation therapy, and immunotherapy are used in the treatment of primary colorectal carcinoma and its more advanced stages (4–9); however, agents with superior antitumor activity are needed to progress the treatment of colorectal cancer. The selective targeting of tumors with monoclonal antibodies (mAb) has emerged as an important therapeutic approach in cancer therapy (7). The antigen target is a critical element in the success of this approach, and one of the most promising targets in colorectal cancer is the A33 antigen: a novel glycoprotein with a molecular weight of 43 kDa, with homology to the immunoglobulin superfamily (10–12). A33 consists of two extracellular immunoglobulin domains, a single transmembrane domain, and a short intracellular tail containing four acylation sites proximal to the transmembrane domain (11, 12). Extensive immunohistochemical analysis of malignant and normal tissues has shown that the antigen is homogeneously expressed by >95% of colon cancers and in the normal intestinal mucosa but not in other epithelial tissues (13, 14). The localization of a murine mAb against the A33 antigen has been studied previously in patients with colorectal carcinoma (15). Phase I trials with I and I murine mAb A33 in colon carcinoma patients showed excellent localization to colorectal cancer and some evidence of tumor response; however, human anti-mouse antibody precluded repeat dosing (15–18). Clinical observations and preclinical data from a radioimmunotherapy model in the nude mouse have also Cancer Therapy: Clinical Authors’Affiliations: Ludwig Institute for Cancer Research, MelbourneTumour Biology Branch; Departments of Nuclear Medicine and Centre for PET, Surgery, Anatomical Pathology, and Medical Physics, Austin Hospital, Melbourne, Victoria, Australia and Ludwig Institute for Cancer Research, New York, New York Received11/14/04; revised1/28/05; accepted 2/1/05. Grant support: National Health and Medical Research Council of Australia grant 98/1905. The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section1734 solely to indicate this fact. Requests for reprints: Andrew M. Scott, TumourTargeting Program, Ludwig Institute for Cancer Research, Level 1, Harold Stokes Building, Austin Hospital, 143-165 Studley Road, Heidelberg,Victoria 3084, Australia. Phone: 61-3-94965876; Fax: 61-3-9496-5892; E-mail: andrew.scott@ludwig.edu.au. F2005 American Association for Cancer Research. www.aacrjournals.org Clin Cancer Res 2005;11(13) July1, 2005 4810 Research. on May 30, 2017. © 2005 American Association for Cancer clincancerres.aacrjournals.org Downloaded from shown that the antitumor effects of radiolabeled mAb A33 can be significantly enhanced by chemotherapeutic agents (19, 20). In view of the long retention time of mAb A33 in tumors, high tumor uptake, and minimal gut toxicity observed in these trials, a humanized version of mAb A33 was constructed to enable repeated dosing without immunogenicity (21). Two phase I trials with huA33 have been conducted, with huA33 alone and huA33 with chemotherapy in patients with colorectal carcinoma (22, 23). Neither of these trials examined the pharmacokinetics of huA33 or the quantitative tumor uptake of huA33. Moreover, the uptake and turnover kinetics in normal bowel of a construct targeting the A33 antigen has not been examined previously. This information is critical in determining the potential therapeutic strategies that can be explored with a humanized construct against this antigen target, in view of the expression of the A33 antigen in this organ. Therefore, to define the targeting characteristics and the serum and colon compartmental kinetics of a humanized form of mAb A33 (huA33), we conducted a phase I, open-label, dose escalation imaging and biopsy-based study of radiolabeled huA33 in patients with colorectal carcinoma, and the results of this study are presented. Materials andMethods huA33 production. The construction, production, and preclinical testing of the humanized CDR-grafted A33 IgG1 (huA33) have been described previously (21–25). This study was conducted according to the Food and Drug Administration (FDA) regulations and as part of an Investigatory New Drug application to the FDA and was also approved by the Human Research Ethics Committee of the Austin Hospital. Trial design. The trial was an open-label, dose escalation, biopsybased phase I study. The primary objectives were to establish the safety of i.v. administered radiolabeled huA33 in patients with colorectal carcinoma and to determine the biodistribution, pharmacokinetics, and immunogenicity of huA33 in these patients. A single infusion of huA33, labeled with [I]huA33 (400 MBq: 10 mCi) and [I]huA33 (40 MBq: 1 mCi), was administered 7 days before scheduled surgery. Each infusion was administered in 100 mL of 5% human serum albumin/normal saline over a 30-minute period. The uptake of radio-iodine in the thyroid and other organs was blocked by saturated solution of KI 10 drops orally thrice daily, commencing before antibody administration and continuing to the day of surgery. Three patients were entered at each of four dose levels of huA33 (0.25, 1, 5, and 10 mg/m). Standard Common Toxicity Criteria were used for evaluation of toxicity. Whole body gamma camera imaging was done on the day of infusion (day 0) and on at least three further occasions up to the day of surgery. Blood samples for pharmacokinetics were obtained before the infusion, immediately after infusion, at 15, 30, 45, and 60 minutes and 2 and 4 hours after infusion, and on three to four further occasions up to the day of surgery. Fig. 1. Four-compartment model representing the kinetics of huA33.The compartments are (1) serum, (2) tissue, (3) colonic epithelium, and (4) tumor. The rate of uptake by the colonic epithelium is k13, whereas the rate of elimination due to colonocyte turnover is k30. Table1. Patient characteristics and sites of disease Patient no. Age (y) Sex Karnofsky performance status at study entry Dose level (mg/m) Prior treatment Sites of disease* Sites of [I]huA33 uptakec 1 63 M 90 0.25 Hemicolectomy, chemotherapy, hepatic lobectomy, chemotherapy Liver metastasis Liver metastasis 2 68 F 90 0.25 Anterior resection, abdominoperineal resection for recurrence Lungmetastasis Lungmetastasis 3 56 M 90 0.25 Anterior resection Liver metastasis Liver metastasis 4 61 M 90 1 Anterior resection, chemotherapy, radiotherapy Liver metastasis Liver metastasis 5 52 F 100 1 Abdominoperineal resection Liver metastasis Liver metastasis 6 66 M 90 1 Hemicolectomy, chemotherapy Liver metastasis Liver metastasis 7 62 M 100 5 Nil Colon tumor Gb 8 48 F 100 5 Hemicolectomy, chemotherapy Liver metastasis Liver metastasis 9 60 F 100 5 Hemicolectomy Liver metastasis Liver metastasis 10 39 F 100 10 Hemicolectomy Liver metastasis Liver metastasis 11 59 M 90 10 Hemicolectomy Liver metastasis Liver metastasis 12 49 F 100 10 Hemicolectomy Liver metastasis Liver metastasis *Determined by CT scan or colonoscopy. cSites of disease identified on planar or single photon emission CT imaging. bNo specific uptake identified at primary tumor sites (note tumor < 1.5 cm size). www.aacrjournals.org Clin Cancer Res 2005;11(13) July1, 2005 4811 Phase I Trial of huA33 in Colorectal Cancer Research. on May 30, 2017. © 2005 American Association for Cancer clincancerres.aacrjournals.org Downloaded from At day 7, patients proceeded to scheduled surgery, where biopsies were obtained to allow assessment of A33 antigen expression, quantitative localization of [I]huA33 to tumor, histologic evaluation, and autoradiographic analysis of [I]huA33 localization in tumor. Patients. Eligibility criteria for entry into the trial were as follows: patients must have histologically proven colorectal cancer; be candidates for surgery for resection of primary and/or metastatic colorectal cancer or candidates for intrahepatic artery pump insertion for liver metastases of colorectal cancer; no treatment with chemoth