Radiolabeling

The DOTA conjugate of hPAM4 was prepared by the sponsor (Immunomedics, Inc.). Quality control testing included using an anti-idiotypic antibody after radiolabeling the DOTA conjugate to ensure immunoreactivity of the final product was maintained. For patient use, radiolabeling of DOTA-conjugated hPAM4 with ¹¹¹InCl₃ (sterile solution; Mallinckrodt/Covidien Imaging Solutions) or ⁹⁰YCl₃ (MDS Nordion) was carried out by a local radiopharmacy by using 5 mg or less protein for ¹¹¹In or 2 mg/mCi or less for ⁹⁰Y, as reported previously (11). Instant thin layer chromatography testing was done and was required to show less than 10% unbound radio-nuclide to be released for patient use. Using a precalibrated dose calibrator, the prescribed radiolabeled doses were prepared as an 8-mL volume in a 10-mL syringe and delivered to the investigational sites for use within 2 hours of preparation.

Treatment

All patients received 3 to 5 mCi of ¹¹¹In-hPAM4 at least 1 week before ⁹⁰Y-hPAM4 to assess PK and ensure that biodistribution and radiation dosimetry estimates for therapy were acceptable. The prepared ¹¹¹In- and ⁹⁰Y-hPAM4 each contained less than 10 mg total antibody and both were administered intravenously by slow injection over 10 minutes. Initially, cohorts of 3 to 6 patients received a single dose of ⁹⁰Y-hPAM4 with ⁹⁰Y doses starting at 15 mCi/m² (555 MBq/m²) and escalated in 5-mCi/m² (185 MBq/m²) increments. DLT was defined as grade 4 hematologic toxicity lasting 7 days or more; a grade 3 nonhematologic toxicity of any duration; or failure of platelets, absolute neutrophil count (ANC), or hemoglobin to recover to grade 1 within 12 weeks, with platelet and red blood cell (RBC) transfusions, as well as growth factors, permitted to aid hematologic recovery. Escalation continued as long as 0 of 3 or 1 of 6 patients at a particular ⁹⁰Y dose encountered DLT, but ceased if there were 2 or more DLTs. The highest ⁹⁰Y dose with 6 patients and at most 1 DLT was determined to be the MTD, with several additional patients then treated at or below the MTD. Prior to each radiolabeled antibody injection, additional unconjugated hPAM4 was administered intravenously over 30 to 40 minutes (10% infused in 10 minutes and if vital signs were stable, remaining dose over 20–30 minutes) during dose escalation (100 mg total antibody doses), but not after the MTD was determined (<10 mg total antibody doses). All antibody administrations were given without steroids or any other premedication.

Study assessments

After ¹¹¹In-hPAM4, blood samples were collected at 0.5, 1, 2, and 4–6 hours and then on days 1, 2, 3, 4–5, and 6–7 for ¹¹¹In radioactivity serum levels which were measured at each site by using a precalibrated g-scintillation counter. Whole-body anterior/posterior planar ¹¹¹In scintigraphy was also carried out repeatedly over 7 days by using a precalibrated g-camera with an ¹¹¹In-calibration standard in the field of view. The initial 6 patients also had total urine collections over 3 days starting 4 hours after administration of ¹¹¹In-hPAM4. All images were evaluated for antibody biodistribution and tumor localization by the local nuclear medicine physician. To proceed to treatment, the biodistribution had to be acceptable with no abnormal pattern of uptake among the major organs that could not be explained by normal variability or incomplete radiolabeling, but demonstrable evidence of tumor targeting was not required. Antibody PK used SAAM II version 1.1.1 software (SAAM Institute, Inc.) to generate multiexponential fits to decay-corrected ¹¹¹In serum levels. The radiation dosimetry methodology has been described previously (12, 13). Briefly a Co-57 patient transmission scan allowed ¹¹¹In counts from organ regions of interest (ROI) on each whole-body image to be corrected for radiation attenuation losses, whereas a computed tomography (CT) image allowed for organ-specific subtraction of background counts from overlapping tissue. Using standard male and female phantom organ masses, solid organ radiation dose estimates were determined by MIRDOSE3.1 software (14) from the geometric mean of corrected anterior and posterior ROI counts, whereas red marrow radiation dose estimates were generated according to the method of Sgouros (15). To proceed with treatment, the dose estimates were required to be below standard limits for external beam radiation therapy (16), but often used for nonmyeloablative radioimmunotherapy trials (<300 cGy for red marrow, <3,000 cGy for liver, and <2,000 cGy for kidney and lung).

Adverse events were classified according to the Med-DRA (Medical Dictionary for Regulatory Affairs, Chantilly, VA) and graded according to the National Cancer Institute Common Toxicity Criteria, version 3.0. Serum chemistries, physical exams, vital signs, and urinalysis were conducted over 12 weeks after treatment, with weekly blood counts or more frequently for grade 3 or higher cytopenias. Blood samples were drawn at baseline and then at 4, 8, and 12 weeks posttreatment for determination of human anti-hPAM4 antibodies (HAHA) by ELISA, using an anti-idiotype antibody as a standard. Values greater than 50 ng/mL were considered positive. Serum levels of CA19-9 were obtained at baseline and then at 1, 4, 8, and 12 weeks posttreatment. Serum levels of the PAM4-reactive mucin were evaluated at the same time points by ELISA assay according to Gold and colleagues (3, 4). CT scans were obtained at baseline and 4, 8 and 12 weeks after ⁹⁰Y-hPAM4, with the best treatment response at any of these time points classified by Response Evaluation Criteria in Solid Tumors (RECIST) criteria (17) as a complete response (CR), partial response (PR), stable, or progressive disease.

Treatment administration

All DOTA-hPAM4 radiolabeled products met release criteria (median unbound isotope: 0.7% ¹¹¹In, 1.1% ⁹⁰Y). During the initial dose escalation portion of the study, 15 patients received ⁹⁰Y-hPAM4 at ⁹⁰Y doses of 15 mCi/m² (555 MBq/m²; n = 4), 20 mCi/m² (740 MBq/m²; n 7), and 25 mCi/m² (925 MBq/m²; n = 4). After the MTD = was determined, 6 additional patients were entered to provide more experience (3 at 15 mCi/m², 2 at 20 mCi/m², 1 withdrawing after ¹¹¹In-hPAM4). All administrations of study drug occurred without interruption or premature termination, with ¹¹¹In- and ⁹⁰Y-hPAM4 both given at total antibody doses of 100 mg for the initial 15 patients and less than 10 mg for the final 6 patients.

Adverse events and dose-limiting toxicity

There were no infusion reactions to administration of study drug. Other than events typically occurring in patients with pancreatic cancer and advanced disease (primarily gastrointestinal complications, pain, and constitutional complaints), the adverse event profile reflected cytopenias that were expected following radioimmunotherapy. The most frequent events (>10% of patients) were vomiting (35%); fatigue, anemia, nausea (30% each); abdominal pain, leukopenia (25% each), constipation, anorexia, thrombocytopenia, diarrhea (20% each), dehydration, back pain, ascites, asthenia, fever, and dyspnea (15% each). Eleven patients had 19 serious adverse events. Of these, 7 events were cytopenias attributed to radio-immunotherapy, 1 event of vomiting was considered possibly related to study drug but with multiple other likely explanations, whereas all other events (dehydration, pain, dyspnea, gastric outlet obstruction, asthenia, confusion) were attributed to complications of underlying disease and were unrelated to study drug. No bleeding episodes occurred, and other than 1 upper respiratory infection and 1 urinary tract infection, none of the patients developed infections. No abnormal pattern of changes occurred in standard serum chemistries, with no grade 3 to 4 events except for several isolated elevations of transaminase, alkaline phosphatase, or bilrubin consistent with hepatic involvement, biliary obstruction, and other abdominal complications expected in this population (data not shown). Grade 3 or 4 anemia was rare at any dose level and required few transfusions of RBCs, but the incidence of severe thrombocytopenia and neutropenia, as well as platelet transfusions and growth factor support, increased with total ⁹⁰Y dose (Table 2). For patients with grade 3 or 4 events, the median time from ⁹⁰Y-hPAM4 therapy to nadir value was 4.8 weeks (range, 3.8–6.6 weeks) for platelets and 4.8 weeks (range, 2.9–5.7 weeks) for neutrophils.

The MTD was declared to be 20 mCi/m², because during dose escalation there were no DLTs at 15 mCi/m², 1 DLT among 7 patients (6 evaluable for DLT assessment, 1 inevaluable after entering hospice 5 weeks posttreatment) at 20 mCi/m², and 3 patients at 25 mCi/m² with DLTs. No DLTs occurred in the 5 patients treated after dose escalation at or below the MTD. All DLTs were due to grade 4 thrombocytopenia and/or neutropenia (thrombocytopenia was generally the more severe) lasting more than 7 days. There was no hematologic DLT due only to prolonged recovery beyond 12 weeks. This was unable to be assessed in 4 patients (3 patients entered hospice with early study terminations prior to recovery and 1 patient had platelet recovery to near grade 1 levels but then developed disseminated intravascular coagulation). Otherwise, the median recovery time from grade 3 or 4 nadirs to grade 1 levels was 2.7 weeks (range, 1.7–4.1 weeks) for platelets and 1.9 weeks (range, 1.0–2.8 weeks) for neutrophils.

Pharmacokinetics and biomarkers

Serum levels of ¹¹¹In-hPAM4, after correction for ¹¹¹In physical decay, generally decreased monoexponentially over 7 days, with a half-life (mean ± SD) of 4.6 ± 0.9 days (range, 3.5–7.0) for the initial 15 patients (100 mg total antibody dose) and 3.8 ± 0.5 days (range, 3.2–4.6) for the final 6 patients (<10 mg total antibody dose). Serum antibody levels were also measured by ELISA assay over 7 days after ¹¹¹In-hPAM4. The median C_max was 21 mg/mL (range, 10–41) with a half-life of 5.1 days (range, 3.9–13.2) for the initial 15 patients, and the median C_max was 4 mg/mL (range, 2–7) for the final 6 patients who had measurable levels over 7 days, but too low for half-life determinations. CA19-9 serum levels were elevated in 18 of the 20 treated patients at study entry. By 4 weeks after ⁹⁰Y-hPAM4, CA19-9 levels had increased more than 25% from baseline in 15 patients and were either stable (n = 3) or had decreased more than 25% (n = 2) in the other 5 patients (2 with no further evaluations, 2 with >25% increases at next evaluation, and 1 with levels still decreased at last evaluation at 12 weeks). In 18 treated patients with measured serum levels of hPAM4-reactive mucin, the median value at study entry was 21 units/mL (range, 1.5–1,398), including 14 patients (78%) with levels above upper limits established in patients without pancreatic cancer (>2.4 units/mL). Posttreatment mucin levels could not be reliably measured due to interference in the ELISA assay from administrated hPAM4.

Biodistribution and tumor targeting

All ¹¹¹In-hPAM4 images were evaluated by the investigators as having normal biodistribution and with no evidence of fixed gastrointestinal or genitourinary uptake. ¹¹¹In-hPAM4 whole-body planar imaging is suboptimal for detecting pancreatic tumors or metastases in the liver. Nonetheless, the investigators reported visual evidence of tumor targeting in 12 patients, including pancreatic tumors or residual tumor in the pancreatic bed (n = 9) or other abdominal sites of confirmed involvement (n = 3). All the visualized pancreatic targeting occurred in patients with the higher serum levels of hPAM4-reactive mucin (28–332 U/mL), and involved 40% (6 of 15) of patients receiving 100 mg total antibody compared with 50% (3 of 6) receiving <10 mg. An internal review by the sponsor of all images found no qualitative differences in biodistribution or intensity of tumor uptake between patients receiving 100 mg or <10 mg total antibody. An example of antibody biodistribution and tumor targeting with ¹¹¹In-hPAM4 imaging is provided in Figure 1.

Open in a separate window

Figure 1

Serial anterior whole-body planar scintigraphic images showing normal antibody biodistribution and tumor targeting in a patient with a recurrent mass in the pancreatic bed after undergoing a Whipple procedure. CT imaging had revealed a 1.5 × 2.1-cm mass surrounding the distal superior mesenteric artery. The images were acquired sequentially over 1 week, beginning 4 hours after administration of ¹¹¹In-hPAM4. Image intensities were normalized by using an ¹¹¹In standard near the left foot to correct for ¹¹¹In physical decay and different acquisition times over this period. Uptake at the pancreatic site is clearly seen at 24 hours (arrow), and becomes progressively more prominent on subsequent images. Mild decreased intensity across the upper aspect of the liver is due to attenuation from breast tissue. Mild focal uptake in the left shoulder and pelvic area is of uncertain significance.

Radiation dosimetry and total body clearance

Table 3 provides estimates of the radiation dose that each mCi of ⁹⁰Y-hPAM4 delivers to the whole body and organs. There are no substantial differences between patients receiving 100 mg or less than 10 mg total antibody, although the liver radiation dose seems modestly decreased in patients receiving additional antibody. Combining the 2 groups, the estimated radiation doses received from treatment are summarized in Table 4. The median dose to the whole body and each individual organ increases with each ⁹⁰Y dose level, as expected. However, even at 25 mCi/m², which exceeds the MTD of 20 mCi/m², the highest radiation dose received by any patient remains well below the defined organ radiation dose limits (red marrow <300 cGy; liver <3,000 cGy, lungs and kidneys <2,000 cGy). Pooled urine was collected in 6 patients at intervals over 3 days after ¹¹¹In-hPAM4 and the counts were corrected for ¹¹¹In-physical decay. The median estimated percent of the administered antibody eliminated in the urine was 3.7% (range, 2.5%–7.5%), with a median of 1.2% excreted by 4 hours, 0.8% from 4 to 24 hours, 0.7% from 24 to 48 hours, and 0.9% over 48 to 72 hours. From this, the median half-life for whole-body clearance of the antibody by urinary excretion was 63 days (range, 33–94 days) compared with 60 days (range, 39–91 days) determined by whole-body scintigraphy for these 6 patients.

Of the 12 patients with visual tumor targeting by ¹¹¹In-hPAM4 scintigraphy, the radiation doses delivered to the primary tumor could be estimated in 2 patients who had discrete focal uptake seen on both anterior and posterior images. The patient shown in Figure 1 was treated at 20 mCi/m², with a 1.5 × 2.1-cm pancreatic mass receiving 5,035 cGy, and remaining stable in size when evaluated 4 weeks later, although new lesions were seen at that time. The other patient was treated at 15 mCi/m², with a much larger 4.3 × 3.4-cm mass in the head of the pancreas receiving a more modest radiation dose of 457 cGy and increasing in size 4 weeks later.

Immunogenicity

Baseline levels of anti-hPAM4 antibodies (HAHA) were all at or below the 50 ng/mL cutoff of the assay. Posttreatment serum samples evaluated for at least 8 weeks in most patients were negative, except for 2 patients with positive levels (one 1,460 ng/mL at 8 weeks and one 1,193 ng/mL at 4 weeks) of uncertain significance, because there were no associated clinical sequelae.

We thank Henry Founds, Preeti Trisal, and Kiril Gordeyev for contributions to the pharmacokinetic and immunoassay analyses; Dion Yeldell for performing radiation dosimetry assessments; and Susan Garl for clinical trial coordination.