Purpose: To determine the in vivo characteristics of huA33, a CDR-grafted humanized antibody against the A33 antigen, we have conducted an open-label, dose escalation, biopsy-based phase I trial of huA33 in patients with colorectal carcinoma.

Experimental Design: Patients with colorectal carcinoma were infused with [131I]huA33 (400 MBq: 10 mCi) and [125I]huA33 (40 MBq: 1 mCi) 1 week before surgery. There were four huA33 dose levels (0.25, 1.0, 5.0, and 10 mg/m2). 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 [131I]huA33 in metastatic colorectal carcinoma. Pharmacokinetic analysis showed no significant difference in terminal half-life (T1/2β) between dose levels (mean ± SD, 86.92 ± 22.12 hours). Modeling of colon uptake of huA33 showed a T1/2 of elimination of 32.4 ± 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 (13). Adjuvant chemotherapy, radiation therapy, and immunotherapy are used in the treatment of primary colorectal carcinoma and its more advanced stages (49); 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 (1012). 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 131I and 125I 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 (1518). Clinical observations and preclinical data from a radioimmunotherapy model in the nude mouse have also 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.

huA33 production. The construction, production, and preclinical testing of the humanized CDR-grafted A33 IgG1 (huA33) have been described previously (2125). 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, biopsy-based 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 [131I]huA33 (400 MBq: 10 mCi) and [125I]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/m2). 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.

At day 7, patients proceeded to scheduled surgery, where biopsies were obtained to allow assessment of A33 antigen expression, quantitative localization of [131I]huA33 to tumor, histologic evaluation, and autoradiographic analysis of [125I]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 chemotherapy, radiotherapy, or immunotherapy for 4 weeks before study entry; ambulatory with a Karnofsky performance status of at least 70%; serum creatinine <0.2 mmol/L; serum bilirubin <20 μmol/L; WBC count >1.5 × 109/L; platelet count >150 × 109/L; prothrombin time <1.3 times upper limit normal; age >18 and <70 years; and ability to provide informed consent. Exclusion criteria included the following: clinically significant cardiac disease (New York Heart Association class III/IV); serious infection requiring treatment with antibiotics or other serious illness; illness requiring the use of steroids or other anti-inflammatory agents; pregnancy or lactation or risk of becoming pregnant; survival expectancy of <6 weeks; evidence of central nervous system tumor involvement; prior administration of mouse mAb or antibody fragment; and positive human anti-mouse antibody titre.

Radiolabeling of huA33. Sterile technique and pyrogen-free glass or plasticware were used in all labeling steps. huA33 was radiolabeled with 131I and 125I as described previously (25). The pooled, radiolabeled huA33 was measured for total radioactivity and then passed through a 0.2 μm filter before administration. Radiochemical purity, immunoreactivity, and in vivo stability of labeled antibodies were analyzed as described previously (25), with a class-matched IgG1 hu3S193 used as a control (26).

Gamma camera imaging/biodistribution. Whole body images of [131I]huA33 biodistribution were obtained in all patients on day 0 after infusion of [131I]huA33 and on at least three further occasions up to day 7 following infusion. Single photon emission computed tomography (CT) images of a region of the body with known tumor were also obtained on at least one occasion during this period. All gamma camera images were acquired on a dual-headed gamma camera (Trionix Research Laboratories, Twinsburg, OH). Whole body images were done as sweeps in a 1,024 × 256–bit matrix, and a standard of known 131I activity was included in the field of view to allow dosimetry calculations.

Pharmacokinetics. Serum obtained from patients following infusion of radiolabeled huA33 was aliquoted in duplicate and counted in a gamma scintillation counter (Packard Instruments, Melbourne, Victoria, Australia). Triplicate standards prepared from the injected material were counted for 131I at each time point with serum samples to enable calculations to be corrected for the isotope physical decay. The results of the serum were expressed as % injected dose per liter (%ID/L).

Pharmacokinetic calculations were done on serum data using a curve-fitting program (WinNonlin, Pharsight Co., Mountain View, CA). A two-compartment model was used to calculate pharmacokinetic variables of Cmax (maximum serum concentration), AUC (area under the serum concentration curve extrapolated to infinite time), CL (total serum clearance), T1/2α and T1/2β (half-lives of the initial and terminal phases of disposition, respectively), A and B (0-time intercepts of the initial and terminal phases of disposition, respectively), and %AUCβ (proportion of AUC under the terminal phase of disposition).

Histologic analysis of biopsies and quantitative tumor uptake. Biopsy specimens from patients were obtained on day 7 after the infusion of radiolabeled huA33. Sections of tumor from surgical specimens were obtained from different areas of the tumor (periphery and central), and portions of each site were allocated for histology, autoradiography, and quantitative measurements. Specimens were examined by an anatomical pathologist in all cases.

The uptake of [131I]huA33 was calculated based on tumor, normal tissue, and standard counts using a gamma scintillation counter and expressed as % injected dose per gram (%ID/g) of tissue. Dry film and emulsion autoradiography of tumor samples was also done from frozen and paraffin sections.

All sections were evaluated for A33 antigen expression on tumor cells. From biopsies, sections of frozen tissue were fixed in acetone, air dried, treated with 0.3% H2O2 for 10 minutes, washed in PBS, and blocked for endogenous biotin and avidin activity. Sections were incubated in 2.5, 5, and 10 μg/mL A33 antibody (Ludwig Institute for Cancer Research, New York, NY) for 30 minutes and following biotin-streptavidin reaction incubated with chromogen 3-amino-9-ethylcarbazole (0.4%, Sigma Chemical Co., St. Louis, MO) and counterstained with Mayer's hematoxylin. Negative controls were included, and results were expressed as percentage of tumor cells expressing A33.

Tumor volume measurements. Measurement of tumor volume was done on CT images in all patients where tumor was identified. Volume measurement was done by drawing regions of interest around visible tumor in each digitized CT slice, and regions of interest area was multiplied by the slice thickness to derive the total tumor volume (cm3). In one patient (patient 7) with primary colonic tumors, tumor volume was obtained by measurement of histologic samples obtained at surgery.

Whole body clearance. Regions of interest were drawn around the body outline of patients on both anterior and posterior whole body images at each imaging time point after infusion of [131I]huA33. Whole body counts were background corrected for 131I decay, and time-activity curves for whole body clearance were then plotted and fitted to a single component exponential clearance expression to calculate the biological whole body clearance half-life (T1/2 biological) of [131I]huA33.

Normal colon compartment kinetics. The amount of huA33 absorbed by the colonic epithelium was calculated from whole body planar gamma camera images, using regions of interest around bowel on sequential images, with application of background and attenuation correction.

The kinetics of the huA33 antibody was represented by a four-compartment model consisting of serum, normal tissue, colonic epithelium, and tumor compartments (Fig. 1). The normal shedding of colonocytes was modeled as an exponential loss from the colon compartment.

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.

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.

Close modal

The amount of antibody in the colonic epithelium is given by:

\[\mathit{q}_{3}(\mathit{t})\ =\ \frac{\mathit{k}_{13}\mathit{A}}{\mathit{k}_{30}{-}{\alpha}}(\mathrm{e}^{{-}{\alpha}\mathit{t}}{-}\mathrm{e}^{{-}\mathit{k}_{30}\mathit{t}})+\frac{\mathit{k}_{13}\mathit{B}}{\mathit{k}_{30}{-}\mathit{{\beta}}}(\mathrm{e}^{{-}\mathit{{\beta}t}}\ {-}\ \mathrm{e}^{{-}\mathit{k}_{30}\mathit{t}})\]

where A, B, α, and β are the usual serum pharmacokinetic variables and k13 and k30 are the rate of uptake and elimination of antibody by colon, respectively. These were estimated by fitting Eq. A to the amount of protein in colon by nonlinear least squares.

Human anti-human antibody measurement. Measurement of immune responses to huA33 in a patient's serum was done using a BIAcore 2000 biosensor (Biacore AB, Uppsala, Sweden), and with an isotype-matched control IgG1 (26), using a method described previously (23, 27). Serum for human anti-human antibody (HAHA) measurement was obtained up to 6 months after infusion of huA33.

Statistical analysis. Statistical analysis was done on pharmacokinetic and tumor localization data obtained from all patients. Any differences in tumor localization, or pharmacokinetics of radiolabeled huA33 ([131I]huA33) in serum, or whole body clearance, between the four dose levels were assessed by a one-way ANOVA. Any correlation between tumor volume and pharmacokinetics, whole body clearance, or mean tumor uptake of [131I]huA33 was done by linear regression analysis. The analysis of uptake and elimination results of huA33 from colon was done using a Kruskal-Wallis rank test.

Patients. Twelve patients (6 males and 6 females) entered and successfully completed the clinical trial. The mean age was 56.9 ± 8.5 years (age range, 39-66 years). The patient demographics, including prior treatment and sites of disease on study entry, are detailed in Table 1. Ten patients had surgery for resection of hepatic metastases or insertion of intrahepatic pump, 1 patient had surgery for resection of lung metastases, and 1 patient with known multiple primary colon tumors underwent surgery for a total colectomy. Surgery was done 7 days after infusion of radiolabeled huA33 in all patients. All patients completed the trial successfully.

Table 1.

Patient characteristics and sites of disease

Patient no.Age (y)SexKarnofsky performance status at study entryDose level (mg/m2)Prior treatmentSites of disease*Sites of [131I]huA33 uptake
63 90 0.25 Hemicolectomy, chemotherapy, hepatic lobectomy, chemotherapy Liver metastasis Liver metastasis 
68 90 0.25 Anterior resection, abdominoperineal resection for recurrence Lung metastasis Lung metastasis 
56 90 0.25 Anterior resection Liver metastasis Liver metastasis 
61 90 Anterior resection, chemotherapy, radiotherapy Liver metastasis Liver metastasis 
52 100 Abdominoperineal resection Liver metastasis Liver metastasis 
66 90 Hemicolectomy, chemotherapy Liver metastasis Liver metastasis 
62 100 Nil Colon tumor  
48 100 Hemicolectomy, chemotherapy Liver metastasis Liver metastasis 
60 100 Hemicolectomy Liver metastasis Liver metastasis 
10 39 100 10 Hemicolectomy Liver metastasis Liver metastasis 
11 59 90 10 Hemicolectomy Liver metastasis Liver metastasis 
12 49 100 10 Hemicolectomy Liver metastasis Liver metastasis 
Patient no.Age (y)SexKarnofsky performance status at study entryDose level (mg/m2)Prior treatmentSites of disease*Sites of [131I]huA33 uptake
63 90 0.25 Hemicolectomy, chemotherapy, hepatic lobectomy, chemotherapy Liver metastasis Liver metastasis 
68 90 0.25 Anterior resection, abdominoperineal resection for recurrence Lung metastasis Lung metastasis 
56 90 0.25 Anterior resection Liver metastasis Liver metastasis 
61 90 Anterior resection, chemotherapy, radiotherapy Liver metastasis Liver metastasis 
52 100 Abdominoperineal resection Liver metastasis Liver metastasis 
66 90 Hemicolectomy, chemotherapy Liver metastasis Liver metastasis 
62 100 Nil Colon tumor  
48 100 Hemicolectomy, chemotherapy Liver metastasis Liver metastasis 
60 100 Hemicolectomy Liver metastasis Liver metastasis 
10 39 100 10 Hemicolectomy Liver metastasis Liver metastasis 
11 59 90 10 Hemicolectomy Liver metastasis Liver metastasis 
12 49 100 10 Hemicolectomy Liver metastasis Liver metastasis 
*

Determined by CT scan or colonoscopy.

Sites of disease identified on planar or single photon emission CT imaging.

No specific uptake identified at primary tumor sites (note tumor < 1.5 cm size).

Adverse events. There were no adverse events related to huA33 observed for any patient entered into the trial. No dose-limiting toxicity was observed, and maximum tolerated dose was not reached.

Radiolabeling and biodistribution of huA33. The mean radiochemical purity of [131I]huA33 and [125I]huA33 was >99% and the mean immunoreactivity was >65% for all infusions. [131I]huA33 and [125I]huA33 were physicochemically stable in serum in vivo up to 7 days after infusion, with retention of high radiochemical purity and immunoreactivity (data not shown).

Biodistribution images of [131I]huA33 showed blood pool distribution only on day 0. There was distinct uptake of [131I]huA33 in sites of metastatic colorectal carcinoma >1.5 cm in all patients, often as early as day 2 after infusion, with localization of [131I]huA33 in metastatic disease in liver and lung sites identified (Fig. 2). Uptake of [131I]huA33 in normal colon was observed in all patients and showed a changing pattern with time, consistent with gradual clearance from bowel.

Fig. 2.

Biodistribution of [131I]huA33 in patient 4. Anterior (A) and posterior (B) whole body gamma camera image 6 days after infusion. Uptake in metastatic lesions in the posterior right lobe of liver (arrows) is clearly seen. Some uptake in normal colon is also evident. C, single photon emission CT transverse image of the liver. D, corresponding CT scan slice.

Fig. 2.

Biodistribution of [131I]huA33 in patient 4. Anterior (A) and posterior (B) whole body gamma camera image 6 days after infusion. Uptake in metastatic lesions in the posterior right lobe of liver (arrows) is clearly seen. Some uptake in normal colon is also evident. C, single photon emission CT transverse image of the liver. D, corresponding CT scan slice.

Close modal

Pharmacokinetics. The serum clearance data of [131I]huA33 are shown in Table 2. The initial and terminal half-lives of huA33 (T1/2α and T1/2β) showed no significant differences between dose levels, with a mean ± SD of 12.74 ± 4.03 hours (P = 0.429) and 86.92 ± 22.12 hours (P = 0.667), respectively. Total serum clearance (CL) also showed no significant differences between dose levels (P = 0.597), with a mean ± SD clearance of 48.56 ± 12.22 mL/h (Table 3). Cmax and AUC increased in a dose-dependent fashion (Table 3). The data fitting yielded two-compartment kinetics, with 86% to 93% of AUC attributable to the elimination phase (Table 3).

Table 2.

Dosage, tumor volume, serum pharmacokinetics, and whole body clearance of [131I]huA33

Patient no.BSA (m2)Dose level (mg/m2)Dose (mg)*Tumor volume (cm3)Serum T1/2α (h)Serum T1/2β (h)WBC§ (h)
1.90 0.25 0.38 270 8.60 69.38 161.48 
1.75 0.25 0.42 37 8.27 75.55 143.92 
2.23 0.25 0.48 48 16.17 102.30 164.69 
1.90 1.45 132 7.25 63.67 131.22 
1.86 1.82 26 11.19 84.24 137.39 
2.12 1.91 2,099 16.77 120.17 140.79 
1.94 6.96 17.98 137.72 176.25 
2.08 8.02 1,770 15.39 78.40 118.58 
2.08 9.60 135 15.26 82.90 126.70 
10 1.60 10 8.83 80 14.97 79.73 143.55 
11 1.86 10 13.62 39 14.07 77.88 149.91 
12 1.55 10 12.76 131 7.00 71.08 132.43 
Patient no.BSA (m2)Dose level (mg/m2)Dose (mg)*Tumor volume (cm3)Serum T1/2α (h)Serum T1/2β (h)WBC§ (h)
1.90 0.25 0.38 270 8.60 69.38 161.48 
1.75 0.25 0.42 37 8.27 75.55 143.92 
2.23 0.25 0.48 48 16.17 102.30 164.69 
1.90 1.45 132 7.25 63.67 131.22 
1.86 1.82 26 11.19 84.24 137.39 
2.12 1.91 2,099 16.77 120.17 140.79 
1.94 6.96 17.98 137.72 176.25 
2.08 8.02 1,770 15.39 78.40 118.58 
2.08 9.60 135 15.26 82.90 126.70 
10 1.60 10 8.83 80 14.97 79.73 143.55 
11 1.86 10 13.62 39 14.07 77.88 149.91 
12 1.55 10 12.76 131 7.00 71.08 132.43 
*

Dose of huA33 infused.

Total volume of tumor identified and measured on CT scan or pathology (patient 7).

Serum pharmacokinetic results from infusion of [131I]huA33.

§

WBC, whole body clearance of [131I]huA33 (T1/2 biological).

Table 3.

Mean (SD) serum pharmacokinetic variables of [131I]huA33 based on total radioactivity

Dose group (mg/m2)nCmax (μg/mL)CL (mL/h)T1/2α (h)T1/2β (h)AUC (μg·h/mL)%AUCβ
0.25 0.12 (0.04) 49.21 (17.52) 11.01 (4.47) 82.41 (17.50) 9.48 (3.48) 92.72 (2.19) 
1.0 0.52 (0.15) 46.30 (16.55) 11.74 (4.78) 89.36 (28.60) 39.27 (9.07) 89.67 (6.69) 
5.0 1.80 (0.26) 58.38 (20.95) 16.20 (1.54) 99.67 (33.02) 151.23 (46.50) 85.94 (1.42) 
10.0 4.03 (0.98) 40.36 (0.17) 12.01 (4.37) 76.23 (4.55) 290.84 (63.49) 90.18 (4.02) 
Dose group (mg/m2)nCmax (μg/mL)CL (mL/h)T1/2α (h)T1/2β (h)AUC (μg·h/mL)%AUCβ
0.25 0.12 (0.04) 49.21 (17.52) 11.01 (4.47) 82.41 (17.50) 9.48 (3.48) 92.72 (2.19) 
1.0 0.52 (0.15) 46.30 (16.55) 11.74 (4.78) 89.36 (28.60) 39.27 (9.07) 89.67 (6.69) 
5.0 1.80 (0.26) 58.38 (20.95) 16.20 (1.54) 99.67 (33.02) 151.23 (46.50) 85.94 (1.42) 
10.0 4.03 (0.98) 40.36 (0.17) 12.01 (4.37) 76.23 (4.55) 290.84 (63.49) 90.18 (4.02) 

Histologic analysis of biopsies. Histologic examination of biopsy specimens showed metastatic colorectal carcinoma in all patient biopsy samples, except for patient 7, who had three primary colon tumors identified in a colectomy specimen. A33 expression was uniform (>90%) in colorectal carcinoma tumor cells in all biopsies examined and negative in all normal tissue biopsied, except for normal colon (patient 7). Tumor volumes ranged from 4 to 2,099 cm3 (Table 2).

Quantitative tumor and normal tissue uptake. Mean quantitative uptake of [131I]huA33 in tumor biopsies ranged from 2.1 × 10−3 to 11.1 × 10−3 %ID/g tumor tissue (Table 4), and individual biopsies reached as high as 14.2 × 10−3 %ID/g (Fig. 3). There was no definite trend observed for localization of [131I]huA33 in tumor between dose levels (Fig. 3) and no significant differences between the dose levels (P = 0.475). In one patient (patient 7), samples of normal terminal ileum and colon were obtained for measurement of [131I]huA33 uptake. The uptake did not substantially differ between bowel regions and was measured as 5.1 ± 2.0 × 10−3 %ID/g tissue (mean ± SD).

Table 4.

Analysis of [131I]huA33 uptake in tumor and normal tissue and relationship to tumor volume

Patient no.Dose level (mg/m2)Tumor volume (cm3)*Site of biopsyTumor uptake (×10−3 %ID/g)T:NT ratio§,T:S ratio
0.25 270 Liver metastasis 4.7 ± 1.9 3.7 ± 1.5 1.9 ± 0.8 
0.25 37 Lung metastasis 2.1 ± 0.5 1.1 ± 0.3 0.5 ± 0.1 
0.25 48 Liver metastasis 3.5 ± 1.1 2.5 ± 0.8 0.8 ± 0.2 
132 Liver metastasis 10.0 ± 3.3 9.1 ± 3.1 2.4 ± 0.8 
26 Liver metastasis 5.0 ± 1.9 5.4 ± 2.1 1.5 ± 0.6 
2,099 Liver metastasis 2.7 ± 0.8 6.4 ± 2.0 2.4 ± 0.8 
Colon primary 5.9 ± 1.0 14.4 ± 2.3 1.5 ± 0.2 
1,770 Liver metastasis 3.4 ± 1.2 5.4 ± 2.0 1.6 ± 0.6 
135 Liver metastasis 4.3 ± 0.8 5.8 ± 1.1 1.5 ± 0.3 
10 10 80 Liver metastasis 2.2 ± 1.8 2.8 ± 2.3 0.5 ± 0.4 
11 10 39 Liver metastasis 11.1 ± 1.9 14.5 ± 2.5 3.4 ± 0.6 
12 10 131 Liver metastasis 5.5 ± 1.8 7.1 ± 2.3 1.2 ± 0.4 
Patient no.Dose level (mg/m2)Tumor volume (cm3)*Site of biopsyTumor uptake (×10−3 %ID/g)T:NT ratio§,T:S ratio
0.25 270 Liver metastasis 4.7 ± 1.9 3.7 ± 1.5 1.9 ± 0.8 
0.25 37 Lung metastasis 2.1 ± 0.5 1.1 ± 0.3 0.5 ± 0.1 
0.25 48 Liver metastasis 3.5 ± 1.1 2.5 ± 0.8 0.8 ± 0.2 
132 Liver metastasis 10.0 ± 3.3 9.1 ± 3.1 2.4 ± 0.8 
26 Liver metastasis 5.0 ± 1.9 5.4 ± 2.1 1.5 ± 0.6 
2,099 Liver metastasis 2.7 ± 0.8 6.4 ± 2.0 2.4 ± 0.8 
Colon primary 5.9 ± 1.0 14.4 ± 2.3 1.5 ± 0.2 
1,770 Liver metastasis 3.4 ± 1.2 5.4 ± 2.0 1.6 ± 0.6 
135 Liver metastasis 4.3 ± 0.8 5.8 ± 1.1 1.5 ± 0.3 
10 10 80 Liver metastasis 2.2 ± 1.8 2.8 ± 2.3 0.5 ± 0.4 
11 10 39 Liver metastasis 11.1 ± 1.9 14.5 ± 2.5 3.4 ± 0.6 
12 10 131 Liver metastasis 5.5 ± 1.8 7.1 ± 2.3 1.2 ± 0.4 
*

Total volume of tumor identified on CT scan or pathology (patient 7).

Site of tumor from which biopsy specimen was obtained for quantitative [131I]huA33 uptake analysis.

Mean ± SD [131I]huA33 uptake from biopsies of central and peripheral portions of tumor sample.

§

T/NT, tumor/normal tissue ratio from surgical tissue samples (mean ± SD).

Normal tissue = liver for all samples, except for patient 7, where normal tissue sample was fat.

T/S, tumor/serum tissue ratio on day of surgery (mean ± SD).

Fig. 3.

Quantitative uptake of [131I]huA33 in (A) tumor biopsies and ratios of (B) tumor/normal tissue (T:NT Ratio) and (C) tumor/serum (T:S Ratio). Points, results from separate biopsy samples from each patient.

Fig. 3.

Quantitative uptake of [131I]huA33 in (A) tumor biopsies and ratios of (B) tumor/normal tissue (T:NT Ratio) and (C) tumor/serum (T:S Ratio). Points, results from separate biopsy samples from each patient.

Close modal

Autoradiography of tumor specimens. Autoradiography showed excellent correlation between localization of radiolabeled huA33 and cellular components of tumor in all biopsies (Fig. 4). There was clear demonstration of penetrance of radiolabeled huA33 to the central portion of metastatic lesions and minimal uptake in necrotic or stromal regions by dry film autoradiography. Emulsion autoradiography showed selective uptake and retention in cellular components of tumor, with minimal uptake in stromal or necrotic areas or normal tissue (e.g., liver; Fig. 4).

Fig. 4.

A, histologic section (H&E; ×1.6) of a metastatic lesion removed from the liver in patient 4 (seen in Fig. 1). B, autoradiograph of huA33 localization in resected hepatic tumor (×1.6). C, high-power (×100) light microscopy view of resected metastatic lesion and adjacent liver. D, dark field autoradiography image of (C), showing white grains corresponding to [131I]huA33 localization to tumor cells within the metastatic lesion and minimal uptake in stroma or normal liver (×100).

Fig. 4.

A, histologic section (H&E; ×1.6) of a metastatic lesion removed from the liver in patient 4 (seen in Fig. 1). B, autoradiograph of huA33 localization in resected hepatic tumor (×1.6). C, high-power (×100) light microscopy view of resected metastatic lesion and adjacent liver. D, dark field autoradiography image of (C), showing white grains corresponding to [131I]huA33 localization to tumor cells within the metastatic lesion and minimal uptake in stroma or normal liver (×100).

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Whole body clearance. The whole body clearance of [131I]huA33 (T1/2 biological) was calculated to be 143.9 ± 16.8 hours (mean ± SD; Table 2). There were no significant differences between dose levels for calculated values (P = 0.766).

Relationship of tumor volume to pharmacokinetics, whole body clearance, and tumor uptake of [131I]huA33. There was no significant difference in pharmacokinetics (serum terminal half-life) of [131I]huA33 in patients with high tumor burden (>1,000 g; n = 2) compared with the remaining patients (P = 0.243). When comparing all dose levels, there was no linear correlation between tumor volume and pharmacokinetics, whole body clearance, or tumor uptake of [131I]huA33.

Normal colon compartment kinetics. The average colon huA33 uptake (k13) half-life was 197 ± 74.6 hours, whereas the average colon elimination (k30) half-life was 32.4 ± 8.1 hours. There were no significant differences in the k13 and k30 half-lives between dose levels (P = 0.22 and 0.32, respectively; Kruskal-Wallis rank test).

Human anti-human antibody measurement. The prestudy BIAcore measurements of a patient's serum showed a mean ± SD of 14.3 ± 2.9 response units (RU), therefore providing a cutoff value for HAHA of 23 RU (mean ± 3 SD). Four of the 12 patients had detectable HAHA following infusion of huA33. These levels were all low, peaking at <90 RU. Patient 6 had a peak measurement of 85.2 RU at 14 days after infusion, patient 7 had a peak RU of 45.2 at 42 days after infusion, patient 8 had a peak at 49.7 RU, and patient 9 had a peak RU of 35.3 at 48 days after infusion of [131I]huA33. Patients 7, 8, and 9 had HAHA measurements returned to normal levels within 3 months; no follow-up values were available for patient 6. No symptoms related to [131I]huA33 infusion, or symptoms of serum sickness, were observed in any patient entered into the study.

This study has shown that radiolabeled huA33 can selectively target primary and metastatic colorectal tumors and penetrate to the center of large necrotic metastatic lesions. Importantly, this study has also shown that huA33 has an elimination T1/2 from bowel consistent with normal colonocyte shedding kinetics (28). The uptake of huA33 in tumor, huA33 pharmacokinetics, and huA33 colon uptake and elimination kinetics were not affected by huA33 protein dose. No adverse events related to a single infusion of huA33 were observed in any patient entered into the study.

Sequential biodistribution images of [131I]huA33 showed high uptake of antibody in sites of metastatic colorectal carcinoma >1.5 cm in all patients, often as early as day 2 after infusion, indicating excellent and rapid tumor uptake (Fig. 2). The intratumoral distribution of [131I]huA33 was also clearly shown by detailed autoradiographic studies of biopsies, with penetrance to central portions of tumor despite the presence of bulky disease and, in some cases, extensive necrosis. This is an important observation, as the barriers to antibody penetrance in tumors due to interstitial pressure, and vascular supply, are often stated to hinder potential efficacy. In the current study, huA33 was clearly able to penetrate to the center of large necrotic tumors to tumors cell nests.

Similar to previous studies with murine A33 mAb, uptake of [131I]huA33 in normal colon was observed in all patients and showed a changing pattern with time, consistent with gradual clearance (1618). The colon compartment kinetics calculations confirmed the elimination T1/2 to be consistent with the turnover time of normal colonocytes (28). There are little published data on the uptake and elimination kinetics of antibodies that target antigens expressed on normal bowel, and this study therefore provides novel data on this important variable. The lack of gastrointestinal symptoms in patients receiving radioimmunotherapy with murine mAb A33 in prior trials is likely to be attributable to this observed colonocyte turnover (transcytosis) in colon mucosa, which reduces the potential cumulative radiation exposure to the gut. The importance of our observation of colonocyte turnover of huA33 is particularly relevant in view of the gastrointestinal toxicity reported with other antibodies that target antigens that are also expressed on normal gut epithelium (29, 30). In addition, the marked differences in retention of [131I]huA33 in normal bowel compared with tumor highlights the importance of the biological properties of cognate antigen in tumor-targeting strategies with recombinant antibodies.

The pharmacokinetics of [131I]huA33 showed a typical serum clearance pattern for IgG, and the 3- to 4-day terminal half-life of [131I]huA33 is at the lower range for published studies of humanized antibodies (3137). The lack of effect of protein dose on pharmacokinetics is an important finding, in view of the small dose therefore required to saturate normal colon (which expresses the A33 antigen and shows uptake of [131I]huA33). This is in marked distinction to other antigen systems (e.g., CD20, epidermal growth factor receptor, and HER-2/neu), where large protein doses are required to saturate the normal tissue antigen pool (33, 34, 3840). In view of the lack of difference in pharmacokinetics, whole body clearance, quantitative tumor uptake, and tumor penetrance between dose levels, further protein dose escalation was not done above 10 mg/m2. The results of this study indicate that 5 to 10 mg/m2 represents an optimal tumor-targeting dosage of huA33. The escalation of huA33 protein dose to define the biological (Fc function) activity of unconjugated huA33 and to further define the toxicity of huA33 at higher dose levels has been explored in separate clinical trials (22, 23).

In published studies, humanized antibodies have shown substantially reduced immunogenicity compared with previous murine antibody trials (27, 2938, 41). Analysis of patient immune responses following radiolabeled huA33 infusion showed low-level HAHA detectable in 4 of 12 patients. In all of these patients, HAHA levels were low (<90 RU), did not increase with time, and, although follow-up data were limited, in three patients quickly fell to the reference range. The biological significance of these findings is uncertain, as all patients received only one infusion of huA33, and changes in pharmacokinetics with repeat infusions were not evaluable.

In conclusion, radiolabeled huA33 when given as a single infusion is safe at the doses used and shows selective and rapid localization to colorectal carcinoma in vivo, with penetration to tumor cell clusters within the center of necrotic tumors. Our study has also shown that huA33 has an elimination T1/2 from normal colon that is consistent with normal colonocyte turnover, which correlates with the lack of gastrointestinal toxicity observed in trials with mAb against the A33 antigen. The excellent targeting characteristics of this humanized antibody indicate clear potential for the targeted therapy of colorectal cancer, and trials of radioimmunotherapy with [131I]huA33, including combination studies of [131I]huA33 with capecitabine, are currently under way.

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 with 18 U.S.C. Section 1734 solely to indicate this fact.

1
Trichopoulos D, Petridou E, Lipworth L, Adami HO. Epidemiology of cancer. In: Rosenberg SA. Cancer: principles and practise of oncology. 5th ed. Philadelphia: Lippincott-Raven; 1997. p. 235–48.
2
Weisburger JH. Causes, relevant mechanisms, and prevention of large bowel cancer.
Semin Oncol
1991
;
18
:
316
–36.
3
Lynch HT, Smyrk T, Watson P, et al. Hereditary colorectal cancer.
Semin Oncol
1991
;
18
:
337
–66.
4
Petrelli N, Douglass HO Jr, Herrera L, et al. The modulation of fluorouracil with leucovorin in metastatic colorectal carcinoma: a prospective randomized phase III trial. Gastrointestinal Tumor Study Group.
J Clin Oncol
1989
;
7
:
1419
–26.
5
Laurie JA, Moertel CG, Fleming TR, et al. Surgical adjuvant therapy of large-bowel carcinoma: an evaluation of levamisole and the combination of levamisole and fluorouracil. The North Central Cancer Treatment Group and the Mayo Clinic.
J Clin Oncol
1989
;
7
:
1447
–56.
6
Mohiuddin M, Marks G. Adjuvant radiation therapy for colon and rectal cancer.
Semin Oncol
1991
;
18
:
411
–20.
7
Scott AM, Cebon J. Clinical promise of tumour immunology.
Lancet
1997
;
349
:
SII19
–22.
8
Douillard JY, Cunningham D, Roth AD, et al. Irinotecan combined with fluorouracil compared with fluorouracil alone as first-line treatment for metastatic colorectal cancer: a multicentre randomised trial.
Lancet
2000
;
355
:
1041
–7.
9
de Gramont A, Figer A, Seymour M, et al. Leucovorin and fluorouracil with or without oxaliplatin as first-line treatment in advanced colorectal cancer.
J Clin Oncol
2000
;
18
:
2938
–47.
10
Catimel B, Ritter G, Welt S, et al. Purification and characterization of a novel restricted antigen expressed by normal and transformed human colonic epithelium.
J Biol Chem
1996
;
271
:
25664
–70.
11
Heath JK, White SJ, Johnstone CN, et al. The human A33 antigen is a transmembrane glycoprotein and a novel member of the immunoglobulin superfamily.
Proc Natl Acad Sci U S A
1997
;
94
:
469
–74.
12
Ritter G, Cohen LS, Nice EC, et al. Characterization of posttranslational modifications of human A33 antigen, a novel palmitoylated surface glycoprotein of human gastrointestinal epithelium.
Biochem Biophys Res Commun
1997
;
236
:
682
–6.
13
Garin-Chesa P, Sakamoto J, Welt S, Real FX, Rettig WJ, Lloyd JO. Organ-specific expression of the colon cancer antigen A33, a cell surface target for antibody-based therapy.
Int J Oncol
1996
;
9
:
465
–71.
14
Van den Eynde BJ, Scott AM. Tumor Antigens. In: Roitt IM, editor. Encyclopedia of immunology. 2nd ed. Vol. 4. London: Academic Press; 1998. p. 2424–31.
15
Welt S, Divgi CR, Real FX, et al. Quantitative analysis of antibody localization in human metastatic colon cancer: a phase I study of monoclonal antibody A33.
J Clin Oncol
1990
;
8
:
1894
–906.
16
Welt S, Divgi CR, Kemeny N, et al. Phase I/II study of iodine 131-labeled monoclonal antibody A33 in patients with advanced colon cancer.
J Clin Oncol
1994
;
12
:
1561
–71.
17
Welt S, Scott AM, Divgi CR, et al. Radio-immunotherapy of colon cancer with monoclonal antibody A33.
Tumor Targeting
1995
;
1
:
299
–300.
18
Welt S, Scott AM, Divgi CR, et al. Phase I/II study of iodine 125-labeled monoclonal antibody A33 in patients with advanced colon cancer.
J Clin Oncol
1996
;
14
:
1787
–97.
19
Scott AM, Welt S. Antibody-based immunological therapies.
Curr Opin Immunol
1997
;
9
:
717
–22.
20
Tschmelitsch J, Barendswaard E, Williams C Jr, et al. Enhanced antitumor activity of combination radioimmunotherapy (131I-labeled monoclonal antibody A33) with chemotherapy (fluorouracil).
Cancer Res
1997
;
57
:
2181
–6.
21
King DJ, Antoniw P, Owens RJ, et al. Preparation and preclinical evaluation of humanised A33 immunoconjugates for radioimmunotherapy.
Br J Cancer
1995
;
72
:
1364
–72.
22
Welt S, Ritter G, Williams C Jr, et al. Preliminary report of a phase I study of combination chemotherapy and humanized A33 antibody immunotherapy in patients with advanced colorectal cancer.
Clin Cancer Res
2003
;
9
:
1347
–53.
23
Welt S, Ritter G, Williams C Jr, et al. Phase I study of anticolon cancer humanized antibody A33.
Clin Cancer Res
2003
;
9
:
1338
–46.
24
Barendswaard EC, Scott AM, Divgi CR, et al. Rapid and specific targeting of monoclonal antibody A33 to a colon cancer xenograft in nude mice.
Int J Oncol
1998
;
12
:
45
–53.
25
Lee FT, Hall C, Rigopoulos A, et al. Immuno-PET of human colon xenograft-bearing BALB/c nude mice using 124I-CDR-grafted humanized A33 monoclonal antibody.
J Nucl Med
2001
;
42
:
764
–9.
26
Scott AM, Geleick D, Rubira M, et al. Construction, production, and characterization of humanized anti-Lewis Y monoclonal antibody 3S193 for targeted immunotherapy of solid tumors.
Cancer Res
2000
;
60
:
3254
–61.
27
Ritter G, Cohen LS, Williams C Jr, Richards EC, Old LJ, Welt S. Serological analysis of human anti-human antibody responses in colon cancer patients treated with repeated doses of humanized monoclonal antibody A33.
Cancer Res
2001
;
61
:
6851
–9.
28
Lipkin M, Sherlock P, Bell B. Cell proliferation kinetics in the gastrointestinal tract of man. II. Cell renewal in stomach, ileum, colon, and rectum.
Gastroenterology
1963
;
45
:
721
–9.
29
Ajani JA, Kelsen DP, Haller D, Hargraves K, Healey D. A multi-institutional phase II study of BMS-182248-01 (BR96-doxorubicin conjugate) administered every 21 days in patients with advanced gastric adenocarcinoma.
Cancer J
2000
;
6
:
78
–81.
30
Knox SJ, Goris ML, Tempero M, et al. Phase II trial of yttrium-90-DOTA-biotin pretargeted by NR-LU-10 antibody/streptavidin in patients with metastatic colon cancer.
Clin Cancer Res
2000
;
6
:
406
–14.
31
Scott AM, Wiseman G, Welt S, et al. A phase I dose-escalation study of sibrotuzumab in patients with advanced or metastatic fibroblast activation protein-positive cancer.
Clin Cancer Res
2003
;
9
:
1639
–47.
32
Posey JA, Ng TC, Yang B, et al. A phase I study of anti-kinase insert domain-containing receptor antibody, IMC-1C11, in patients with liver metastases from colorectal carcinoma.
Clin Cancer Res
2003
;
9
:
1323
–32.
33
Baselga J, Tripathy D, Mendelsohn J, et al. Phase II study of weekly intravenous recombinant humanized anti-p185HER2 monoclonal antibody in patients with HER2/neu-overexpressing metastatic breast cancer.
J Clin Oncol
1996
;
14
:
737
–44.
34
Pegram MD, Lipton A, Hayes DF, et al. Phase II study of receptor-enhanced chemosensitivity using recombinant humanized anti-p185HER2/neu monoclonal antibody plus cisplatin in patients with HER2/neu-overexpressing metastatic breast cancer refractory to chemotherapy treatment.
J Clin Oncol
1998
;
16
:
2659
–71.
35
Caron PC, Jurcic JG, Scott AM, et al. A phase 1B trial of humanized monoclonal antibody M195 (anti-CD33) in myeloid leukemia: specific targeting without immunogenicity.
Blood
1994
;
83
:
1760
–8.
36
van Hof AC, Molthoff CF, Davies Q, et al. Biodistribution of (111)indium-labeled engineered human antibody CTMO1 in ovarian cancer patients: influence of protein dose.
Cancer Res
1996
;
56
:
5179
–85.
37
Scott AM, Lee FT, Hopkins W, et al. Specific targeting, biodistribution, and lack of immunogenicity of chimeric anti-GD3 monoclonal antibody KM871 in patients with metastatic melanoma: results of a phase I trial.
J Clin Oncol
2001
;
19
:
3976
–87.
38
Baselga J, Pfister D, Cooper MR, et al. Phase I studies of anti-epidermal growth factor receptor chimeric antibody C225 alone and in combination with cisplatin.
J Clin Oncol
2000
;
18
:
904
–14.
39
Kaminski MS, Zasadny KR, Francis IR, et al. Radioimmunotherapy of B-cell lymphoma with [131I]anti-B1 (anti-CD20) antibody.
N Engl J Med
1993
;
329
:
459
–65.
40
Press OW, Eary JF, Appelbaum FR, et al. Phase II trial of 131I-B1 (anti-CD20) antibody therapy with autologous stem cell transplantation for relapsed B cell lymphomas.
Lancet
1995
;
346
:
336
–40.
41
Clark M. Antibody humanization: a case of the “Emperor's new clothes”?
Immunol Today
2000
;
21
:
397
–402.