Phase I Clinical Trial of the Chimeric Monoclonal Antibody (C30.6) in Patients with Metastatic Colorectal Cancer1

4674 Vol. 6, 4674–4683, December 2000 Clinical Cancer Research

Phase I Clinical Trial of the Chimeric Monoclonal Antibody (c30.6) in Patients with Metastatic Colorectal Cancer1

Robyn L. Ward,2 Deborah Packham, uptake. At the 50 mg/m2 level the half-life and maximum -Anne M. Smythe, Jayne Murray, serum concentration were 81 ؎ 15 h and 7.9 ␮g/ml, respec Peter Anderson-Stewart, Neil Kitchen, tively. One patient developed a low-level human anti-c30.6 response. Tumor response was assessed by computed tomog- Rosyln Muirhead, Peter Phillips, Peter Gray, raphy, positron emission tomography scanning, and serial Grant Bigg-Wither, Krishnan Prabakaran, carcinoembryonic antigen measurements. There were no Judy Freund, Michael Fullham, Michele Rule, partial responses, although positron emission tomography David Dalley, Alan Meagher, scanning demonstrated some reduction in tumor activity in Nicholas J. Hawkins, and Glenn M. Smith three individuals. The chimerized c30.6 antibody is not immunogenic in Cooperative Research Centre for Biopharmaceutical Research Ltd., Darlinghurst 2010, New South Wales [R. L. W., D. P., A. M. S., P. A- humans and appears worthy of further study. It does, how- S., N. K., R. M., P. P., G. M. S.]; Departments of Medical Oncology ever, produce a unique profile of side effects that can be well [R. L. W., M. R., D. D.], Nuclear Medicine [K. P., J. S.], Colorectal controlled with premedication. Surgery [A. M.], and Radiology [G. B-W.], St. Vincent’s Hospital, Darlinghurst 2010, New South Wales; Schools of Biotechnology [N. K., P. P., P. G., G. M. S.] and Pathology [R. L. W., N. J. H.], INTRODUCTION University of New South Wales, Sydney 2052, New South Wales; Over the last few years, a number of clinical studies have and Department of Nuclear Medicine [M. F.], Royal Prince Alfred provided evidence that monoclonal antibodies are of value in the Hospital, Sydney 2050, New South Wales, Australia treatment of a variety of cancers, including breast, lymphoma and colorectal tumors (1–3).3,4 Although adjuvant chemother- ABSTRACT apy modestly improves the disease-free and overall survival of patients with Dukes stage C colorectal cancer, there is a need for The murine antibody 30.6 recognizes an antigen that is new adjuvant therapies that have both improved efficacy and expressed on a high proportion of colorectal carcinomas and fewer side effects. In this regard, the proven efficacy of adjuvant their metastases. We report the results of single-dose esca- 17-1A therapy in a small group of patients with Dukes stage C lation studies of the chimeric 30.6 (c30.6) monoclonal anti- colon cancer has shown that antibodies may have optimal effi- body in metastatic colorectal cancer, to evaluate its safety, cacy when used in the setting of minimal residual disease (2). pharmacokinetics, and biodistribution. Furthermore, there is a good rationale for the use of combina- Recombinant c30.6 (IgG1␬) antibody was secreted tions of antibodies or indeed for their use with traditional from Chinese hamster ovary cells and purified by a multi- chemotherapy, so as to deliver a range of potentially synergistic step chromatography process. Seventeen patients with met- antitumor activities (4–6). astatic colorectal cancer were enrolled in this dose escalation The murine monoclonal antibody 30.6 recognizes an anti- study. The first four patients were treated with 3 mg of 123 gen that is expressed on colorectal carcinomas and their metas- I-labeled c30.6, whereas the next 13 received a single dose 2 tases. Expression of the antigen is greatest on well-differentiated of unlabeled antibody (maximum dose, 50 mg/m ). colorectal adenocarcinomas, is less pronounced on poorly dif- The most frequent side effect was a novel syndrome of ferentiated adenocarcinomas, and is usually absent from most severe burning and erythema of the face, chest, neck, ears, undifferentiated carcinomas (7). The biochemical nature of the palms, soles, and genitalia. The frequency of this syndrome 30.6 antigen has not been elucidated, but it is expressed only on was markedly reduced in those patients premedicated with the luminal surface of glandular cells and is not released into the high doses of histamine receptor 1 and histamine receptor 2 circulation. The antigen is found in gastrointestinal epithelium blockers. Other side effects were mild and predictable. Bio- as well as in pancreatic acini, hepatocytes, alveolar pneumo- distribution studies showed a rapid and intensive hepatic cytes, and prostatic acinar epithelium. However, it is not expressed in other organs or tissues of the urogenital tract or central nervous system (7). The murine 30.6 antibody has been shown to localize to s.c. human colorectal cancer xenografts in Received 6/6/00; revised 9/15/00; accepted 9/18/00. The costs of publication of this article were defrayed in part by the nude mice (8) as well as to primary and secondary tumor payment of page charges. This article must therefore be hereby marked deposits in patients with metastatic colorectal cancer (8, 9). advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by the Australian Government Cooperative Research Pro- gram. 2 To whom requests for reprints should be addressed, at Department of 3 Internet address for Rituxan full prescribing information, 2000: http:// Medical Oncology, St. Vincent’s Hospital, Victoria Street; Darlinghurst www.genentech.com/Medicines/rituxan_insert.html. 2010, New South Wales, Australia. Phone: 61292958412; Fax: 4 Internet address for healthcare professionals prescribing information 61283823386; E-mail: [email protected]. for Herceptin, 2000: http://www.herceptin.com/prof/pi000.html.

Furthermore, the antibody, whether used alone, radiolabeled, or chromatography and then treated with solvent/detergent to in- conjugated to cytotoxic drugs, is able to strongly inhibit the activate lipid-enveloped viruses. Additional purification by an- growth of human colorectal carcinoma xenografts in nude mice ion and cation exchange chromatography removed residual pro- (10). Phase I clinical studies of N-acetylmelphalan coupled to teins, solvent/detergents, and nucleic acids. The purified c30.6 30.6 showed that patients produced a HAMA5 that precluded was additionally purified and formulated into 0.9% saline using additional dosage escalation (11, 12). two gel filtration columns. Finally, the formulated bulk was The development of HAMA could impair the therapeutic sterile- and viral-filtered before dispensing into Hypak glass effectiveness of 30.6, either by interfering with its binding to syringes (Becton Dickinson, Lincoln Park, NJ). Stability studies antigen or by reducing its bioavailability. Replacing the murine showed that the c30.6 antibody was stable in this formulation for constant regions with a human ␥1 region was likely to reduce at least 16 months. Purity, identity, activity, and safety were the immunogenicity of the antibody and also maximize its confirmed before release of the vialed product for clinical use. effector function. Mount et al. described a chimeric version of Radiolabeling of c30.6. The c30.6 antibody (5 mg in 2 ϭ 123 this antibody (c30.6) that binds with moderate affinity (Ka ml) was labeled with I (2 GBq in 1 ml; ARI, Sydney) using 8 Ϫ1 1 ϫ 10 M ) to its antigen and can mediate in vitro ADCC (13). the Iodogen method, and the labeled antibody was purified by Although this chimeric antibody was not able to lyse cells in the gel filtration using a Superose 12 HR column (Pharmacia, presence of either human or rabbit complement, it had antitumor Uppsala, Sweden) coupled with fast protein liquid chromatog- activity in mice with SCID bearing s.c. human colorectal cancer raphy. Before purification, the iodine incorporation was 68.0 Ϯ xenografts. A 40% reduction in tumor size was observed after 7.0%. The immunoreactive fraction of the labeled antibody was i.p. c30.6 administration, with maximal antitumor activity while estimated by the Lindmo method (14) using the human colorec- the c30.6 antibody was being administered. tal cell line HT-29. The average immunoreactivity of the radio- The chimerized 30.6 antibody, therefore, has a number of labeled preparations was 60.7 Ϯ 8.6%. The radiolabeling characteristics that make it an attractive antibody for additional method was validated to produce sterile and pyrogen-free prod- clinical development. These include its ability to induce ADCC, uct. Serum samples from patients injected with radiolabeled its pattern of tissue reactivity, affinity, potential for reduced antibody were analyzed by gel filtration chromatography for the immunogenicity, and documented antitumor activity in animal presence of aggregates and free iodide using a Superose 12 models. We report here the results of single-dose escalation HR10/30 gel filtration column. studies of the chimeric 30.6 monoclonal antibody in patients Patient Selection. For entry to the study, the patients with metastatic colorectal cancer. The primary objectives of the were required to have histologically proven metastatic colorec- study were to evaluate safety, pharmacokinetics, and biodistri- tal cancer and to have adequate renal (creatinine Ͻ125% of the bution of the c30.6 antibody in this patient population. upper limit of the normal range), hepatic (bilirubin Ͻ125% of the upper limit of the normal range; prothrombin time Ͻ 1.3 PATIENTS AND METHODS times control), and hematological function (white blood count Ͼ ϫ 9 Ͼ ϫ 9 Production of c30.6. Recombinant c30.6 (IgG1␬) anti- 4.0 10 /liter; platelet count 100 10 /liter; hemoglobin Ͼ body is secreted from CHO cells. The cloned 30.6 antibody 100 g/liter) as well as the presence of measurable metastatic Ն heavy- and light-chain, variable region cDNAs (obtained from disease (at least one site 1 cm). Each patient was also required Ͻ the Austin Research Institute, Melbourne) were subcloned into to have a WHO performance status of 2 and a life expectancy the antibody expression vectors pG1D102 and pKN100 (Med- of at least 3 months. Patients were excluded if they had under- ical Research Council, Cambridge, United Kingdom). The vec- gone chemotherapy or radiotherapy in the preceding 4 weeks or tors containing heavy and light chains were transfected into the had received immunosuppressive therapy in the preceding 3 host cell line CHO DG44 (from Dr. Larry Chasin, Columbia months. University, New York). After selection and screening, the pro- The 30.6 antigen is not detectable in paraffin embedded duction cell line 10A75H2.2F5 was isolated. The master cell tissues, but is expressed on almost all of the moderately and bank of 10A75H2.2F5 was found to be free of microbial con- most of the poorly differentiated adenocarcinomas of the colo- taminants, murine adventitious virus, and retrovirus. As ex- rectum (7), and therefore documentation of antigen expression pected, transmission electron microscopy demonstrated the was not required for entry into the study. Patients with severe presence of endogenous retrovirus particles in the CHO cells. nonmalignant systemic disease or who were HIV positive or had c30.6 antibody was manufactured using batch fermentation with uncontrolled infection were precluded from entry. Those indi- serum-free media and was purified via a multistep procedure viduals who had previously been exposed to murine or chimeric incorporating five chromatography and two viral inactivation/ antibody or antibody fragments were also excluded from entry. removal steps. The antibody was separated by Protein A affinity Detailed informed consent was obtained from all of the patients in accordance with the St. Vincent’s Hospital Human Ethics committee. Clinical Trial Design. The first four patients entered in this study received 3 mg 123I-labeled c30.6 (30 mCi). The next 5 The abbreviations used are: HAMA, human antimouse antibody re- 13 patients received a single dose of antibody at doses of 10 (5 sponse; ADCC, antibody-dependent, cellular cytotoxicity; SCID, severe patients), 25 (5 patients), and 50 mg/m2 (3 patients). Immedi- combined immunodeficiency; CHO, Chinese hamster ovary; HACA, ately after the infusion of unlabeled antibody, 6 of the 13 human anti-c30.6 antibody; CEA, carcinoembryonic antigen; CT, com- 123 puted tomography; PET, positron emission tomography; SPECT, single- patients also received 3 mg I-labeled c30.6 antibody. Patients photon emission computed tomography; H1, histamine receptor 1. were selected for administration of 123I-labeled c30.6 on the

basis of the site and extent of their disease as well as perform- ard curves and serum antibody concentrations were calculated ance status. Those individuals who received radiolabeled anti- using the Assay-Zap assay program (Elsevier-Biosoft, Cam- body were treated with Lugol’s iodine for 3 days before and 2 bridge, United Kingdom). The detection limit of the assay was days after the antibody infusion. 20 ng/ml, although c30.6 levels of 1 ng/ml were detectable. Study Measurements. All of the adverse events that Pharmacokinetic data analysis was performed using the nonlin- occurred within 28 days of administration of the antibody were ear parameter estimation application Minim (Dr. Robert Purves, recorded and graded according to the Southwest Oncology University of Otago Medical School, Dunedin, New Zealand). Group Criteria. Measurement of hematology, serum chemistry, Measurement of HACA Levels. HACA levels were deliver function tests, complement, HACA, and serum c30.6 levels termined using a bridging ELISA. In this assay, serum HACAs were performed at regular intervals for a period of 6 months act as a bridge between c30.6 antibody coated on an ELISA after the treatment. Physical examination, serum CEA levels, plate and biotinylated c30.6 (sulfo-NHS-LC-biotin; Pierce) in CT and PET imaging studies were performed at 4 to 6 weeks solution. A signal, therefore, indicates that biotin has been after treatment to assess the tumor response. A partial response linked to the solid phase and that anti-c30.6 antibodies were in was defined as a decrease of Ͼ50% in the total sum of the the serum sample. Plates were coated with c30.6 for 16 h at 4°C products of the bidimensional measurements. Complete remis- (2 ␮g/ml in coating buffer) and blocked as described above. sion was defined as the disappearance of all of the disease, After the addition of biotinylated c30.6, patient serum samples whereas stable disease was defined as no significant change in were added to the plate. Each plate also included the following tumor measurements. A single expert (G. B-W.) who was un- controls: goat antihuman IgG Fc␥ antibody (0.1–1000 ng/ml; aware of the treatment protocol examined all of the imaging Jackson ImmunoResearch) for quantitation of the HACA re- studies. sponse, sheep anti-c30.6 as a positive control and patient base- Pharmacokinetics of c30.6. To determine the pharma- line serum samples as negative controls. Alkaline phosphatase- cokinetics of c30.6, serial blood samples were assayed by either streptavidin was added to washed plates, and color was gamma counting to detect radiolabeled c30.6 (used in the first developed and analyzed as described above. The limit of detec- four study patients) or ELISA to detect both unlabeled and tion of this assay was 2 ng/ml of goat antihuman IgG Fc␥. radiolabeled antibody (used in all other study patients). In each Imaging and Dosimetry Studies. In those patients who patient who received radiolabeled antibody, EDT- treated blood received 123I-labeled c30.6, anterior and posterior whole-body samples were collected at 2, 4, 8, 15, and 30 min and 1, 4, 24, planar images (256 ϫ 1024 matrix, 15 min/m) were performed and 48 h. After 48 h 1-ml plasma samples were counted on a immediately and at 1, 24, and 48 h after injection. In addition gamma counter (autogamma 5650; Packard Instruments, SPECT images (128 ϫ 128 matrix, elliptical orbit, 64 views, Downers Grove, IL). The total administered dose was deter- 360°, 30 s/view) of the chest and abdomen were obtained at 24 mined using a dose calibrator, and the disintegrations per and 48 h. A GE XRT gamma camera and high-resolution, minute were calculated by correcting for the efficiency of the low-energy collimator was used for all of the imaging. Radiation gamma counting. dosimetry was estimated by using the whole-body images from Serum c30.6 levels were determined using a competitive, 0, 4, 24, and 48 h after injection. The cumulated activity was solid-phase enzyme immunoassay, in which chimeric antibody calculated for various organs of interest and then converted into in serum competes with exogenous biotin-labeled c30.6 for residence times. These times were entered in to the MIRDOSE binding to sheep anti-c30.6 antibodies coated onto ELISA program (Oak Ridge National Laboratories) to determine the plates. Plates (Nunc-Immuno MaxiSorp) were incubated over- internal radiation dosimetry. night at 4°C with affinity-purified sheep anti-c30.6 antibody diluted 1:200 in coating buffer (100 mM sodium bicarbonate, 0.1% Bronidox, pH 8.5). After two washes in PBS/1% Tween/ RESULTS 0.1% Bronidox, the plate was blocked with PBS/1% skim milk/ Patient Characteristics. The characteristics of the 17 0.5% BSA/0.1% Bronidox for 90 min at 37°C. patients with progressive metastatic colorectal cancer (7 rectal, Patient serum samples were collected before the infusion, 10 colon) who entered this study are shown in Table 1, along at 1, 4, 24, and 48 h, and again at 6, 8, and 15 days. Samples and with a summary of prior therapies and sites of disease at the time standards were heat-inactivated (60°C, 30 min), diluted in of administration of c30.6. The participants were aged from 37 pooled heat-inactivated human serum, and then mixed with an to 78 years, and the sites of their metastatic disease included the equal volume of biotinylated c30.6 (1:2000 in blocking buffer liver (n ϭ 16), lungs (n ϭ 5), mesentery (n ϭ 2), lymph nodes diluted in PBS/0.1% skim milk/0.05% BSA/0.1% Bronidox). (n ϭ 5), bones (n ϭ 3), adrenal (n ϭ 1), and pelvis (n ϭ 2). This mixture was incubated on the washed plate at 37°C for 2 h, Seven individuals had initially presented with metastatic dis- washed, and then incubated with alkaline phosphatase-strepta- ease, and in two of these cases the primary tumor had not been vidin (1:2000 in sample diluent; Jackson ImmunoResearch Lab- resected before antibody administration. Of the 10 individuals oratories, West Grove, PA) for 60 min at 37°C. After additional without metastatic disease at initial presentation, 5 had received washes, p-nitrophenyl phosphate in carbonate buffer was added, adjuvant 5-fluorouracil-based chemotherapy shortly after resec- and the plate was incubated at 37°C until the lowest point on the tion of their primary tumor, and one of these individuals devel- standard curve (1 ng/ml c30.6) had an absorbance at 410 nm of oped metastatic disease while undergoing adjuvant therapy. 2.0 (MR 7000 Microplate Reader; Dynatech). Each assay in- At the time of antibody therapy, chemotherapy was con- cluded a set of standards of c30.6 (2–1000 ng/ml) diluted in sidered inappropriate in five patients, either because of progres- pooled human serum and mixed with biotinylated c30.6. Stand- sive disease despite palliative therapy (n ϭ 3), relapse on

Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2000 American Association for Cancer Research. Downloaded from Table 1 Clinical details of all patient receiving c30.6 in this study Patient Sites of Sex, Dose Primary Primary Metastases at Prior Prior Prior disease at time of Response determined Patient Age (mg) site resected presentation surgerya radiotherapya chemotherapya c30.6 treatmentb by CT/PET scan 1 37, M 2.1c SCd Y Y LAR (3 wk) Nil Nil Multiple bilobar hepatic NA, withdrew from study 2 78, M 3.1c DC Y N LH (3 yr) Nil Adjuvant 5FU/FA (3 yr) Left cervical lymph node, 6 lesions P, 2 NC hepatic (10–40 mm), pulmonary, mesentery, right

clincancerres.aacrjournals.org adrenal 3 57, M 3.2c R Y N LAR (3 yr) Nil Nil 1 right hepatic lobe (20 mm), 2 new lesions, 2 P, 2 NC nodes at porta hepatis and around duodenum 4 58, M 3.1c TC Y Y STC (6 mo) Nil Nil Bilobar hepatic (10–15 mm) 2 new lesions, 4 P, 2 NC and left femur 5 67, M 17 R Y N AR (4 yr) Adjuvant pelvis (4 yr) Adjuvant 5FU/FA (4 yr) 2 hepatic metastases (8 & 60 2P mm) 6 52, M 17, 3.9c AC Y N RH (9 mo) Nil Adjuvant 5FU/FA (2 mo) Right hepatic metastases (20– ND (died at 8 wk) 50 mm) 7 51, F 13.8, 3.0c R N Y ND Nil Nil Bilobar hepatic metastases 3 hepatic lesions, NC (10–20 mm), rectal tumor infiltrating prerectal space 8 56, M 16, 3.0c R Y N AR (5 yr); AP (4 yr) Palliative pelvis (2 yr); brachy- Palliative i.a. 5FU/Mito (18 Bony, pelvis, pulmonary On CT, NC on PET, P in lung

on September 27, 2021. © 2000American Association for Cancer therapy (2 yr); palliative left mo); palliative i.v. 5FU/ but response in pelvis iliac crest (9 mo) Mito/FA Research. (6 mo); camptosar (8 wk) 9 72, M 13.8 R Y N AR (4.5 yr) Adjuvant pelvis (4.5 yr) Adjuvant 5FU/FA (4 yr) Right hilum, para-aortic lymph ND, refused follow up CT nodes, hepatic 10 69, M 40 R Y N LAR (6 yr) Palliative, local recurrence Palliative, radiosensitizer 5FU Left hepatic, pelvis, pulmonary 1 new pulmonary lesion, 2 P (3.5 yr) (3.5 yr) (10–40 mm) in lung 11 69, M 45, 3.0c DC N Y ND Nil Nil Bilobar hepatic metastases (5– Slight P hepatic lesions, 30 mm), descending colon response in primary tumor 12 75, M 47.5 SC Y N PC (18 mo) Nil Nil Bilobar hepatic, Bony L2,T10 , ND (died at 18 days) left iliac crest 13 42, M 48, 3.0c AC Y Y RH (2 yr) Palliative mediastinum (3 mo) Palliative 5FU/FA (18 mo); Hepatic (up to 80 mm), 4 NC (lung, liver, lymph palliative 5FU (3 mo) pulmonary (2 mm), nodes) paraortic LNs (up to Differential response on PET 20 mm) 14 73, M 47.5 SC Y N AR (18 mo) Nil Adjuvant 5FU/FA (1 yr) Hepatic (2–30 mm) 2 lesions P, 2 NC 15 56, M 95, 3.0c AC Y N RH (4 yr); Local Nil Palliative 5FU (3 mo) Bilobar hepatic and omental CT scan ND; suggestion of wound metastases disease early response on PET (6 mo) 16 70, M 95 HF Y Y RH (3 mo) Nil Nil Bilobar hepatic metastases 2 hepatic lesions P, 2 hepatic (15–30 mm), portocaval lesions NC; lymph node NC

node (20 mm) Research Cancer Clinical 17 63, M 90 R Y Y AP (4 mo) Adjuvant pelvic (2 mo) Radiosensitizer 5FU (2 mo) Bilobar hepatic metastases CT scan ND, P on PET (5–20 mm) a Time since last treatment in parentheses. b Size range in parentheses. c Dose in milligrams of radiolabeled c30.6 administered after unlabeled antibody. d SC, sigmoid colon; R, rectum; DC, descending colon; TC, transverse colon; HF, hepatic flexure; AC, ascending colon; AP, abdominoperineal resection; RH, right hemicolectomy; PC, protocolectomy; AR, anterior resection; LAR, low anterior resection; STC, subtotal colectomy; LH, left hemicolectomy; ND, not done; 5FU, 5-fluorouracil; FA, folinic acid; i.a., intraarterial; Mito, mitomycin C; NA, not assessable; NC, no change; P, progressed. 4677 4678 c30.6 Antibody for the Treatment of Colon Cancer

Table 2 Acute adverse events related to infusion of the c30.6 antibody An acute event was defined as a side effect occurring within 24 h after treatment with the antibody. Those events that were either definitely or possibly a consequence of antibody administration are included in the table. Dose (mg/m2)

Radiolabeled 10 25 50 Side effect (na ϭ 4) (n ϭ 5) (n ϭ 5) (n ϭ 3) Generalized Back pain 0 0 1 0 Headache 1 0 0 0 Rigors and/or temperature 1 0 2 1 Shoulder/hip pain 0 1 0 1 Gastrointestinal Abdominal pain 0 0 0 3 Nausea 0 1 1 0 Nausea and vomiting 0 1 0 2 Respiratory Breathless 0 0 1 0 Sneezing 0 0 1 2 Mucocutaneous Burning erythema (eyes, external auditory canal, nasal mucosa) 0 3 (2ML,b 1S) 4 (1ML, 2MD, 1S) 2 ML Burning erythema (palms, soles) 0 3 (1ML, 2MD) 4 (1ML, 2MD, 1S) 2 ML Burning (genital) 0 2 (1ML, 1MD) 2 (1ML, 1S) 1 ML Burning erythema (face, ears) 0 2 (1MD, 1S) 3 (1ML, 1MD, 1S) 2 ML Rash 0 0 0 1 ML Tumor pain Chest 0 1 0 0 Right hip 0 0 1 0 Hepatic 0 0 1 0 Sciatica 0 1 0 0 Total adverse events 2 15 21 17 a n is patients per group. b ML, mild; MD, moderate; S, severe.

adjuvant therapy (n ϭ 1), or refusal to undertake palliative external auditory canal, injection of nasal mucosa, stuffy nose, therapy (n ϭ 1). An additional 12 individuals had progressive and discomfort or burning around the lips and throat. These disease but were as yet either asymptomatic or their symptoms symptoms typically began 30–300 min after the infusion com- were well controlled with other medications. menced (median, 70 min) and lasted from 5 min to 4.3 h Antibody Infusions. The unlabeled c30.6 antibody was (median, 1.5 h). Although each patient did not invariably expe- administered in an outpatient clinic by infusion through a pe- rience burning erythema at each site, the temporal involvement ripheral line. Dose levels of 10, 25, and 50 mg/m2 were diluted of the skin typically followed the following sequence: face/ in 50, 100, or 500 ml 0.9% sodium chloride and delivered over genitalia, palms, and then soles. These symptoms were self- 40, 60, or 120 min, respectively. Ten individuals were injected limiting and did not require admission to hospital, yet in some with a slow i.v. push of 123I-labeled c30.6 (30 mCi), either alone patients they were of such severity as to require narcotic anal- (patients 1–4) or immediately after the completion of the unla- gesia. Once these symptoms had begun, they were refractory to beled antibody infusion (patients 6–8, 11, 13, 15; Table 1). The all of the modalities of treatment, including cold packs, drugs first seven patients (1–7) did not receive a premedication before such as H1 blockers (promethazine, 25–50 mg i.v.), hydrocor- the treatment, whereas the next seven (8–14) were premedicated tisone (up to 200 mg iv), Sudafed (60 mg p.o.), paracetamol, and with a combination of loratidine (5–10 mg) and paracetamol narcotics. Unfortunately, the severity of pain meant that skin (500 mg) up to 1 h before antibody administration. The final biopsies of affected sites could not be performed. At the dose three patients (15–17) were premedicated with promethazine level of 10 mg/m2 all 5 patients experienced these mucocuta- (50–75 mg i.m.) 2 h before antibody infusion and with raniti- neous side effects, and of the 10 events, 2 were mild, 6 were dine (50 mg i.v.) 1 h before treatment. moderate, and 2 were severe. At the 25-mg/m2 level, four of the Antibody Toxicity. All but one patient completed the five patients demonstrated this side effect, with four mild, five scheduled infusion of antibody. In this patient the last 2 of 48 moderate, and four severe reactions. mg of antibody were not administered because he experienced These reactions occurred despite pretreatment in some uncontrolled, severe burning erythema of his face, palms, soles, cases with loratidine and paracetamol 1 h before infusion (2 of and genitalia. A total of 56 adverse events occurred in the first the 5 individuals at the 10-mg/m2 dose level and all of the 24 h after infusion of antibody, and of these 55 were directly individuals at the 25-mg/m2dose level). At the of 50-mg/m2 attributable to the antibody infusion (Table 2). The most fre- dose level premedication with promethazine and ranitidine was quent side effect (56% of all of the adverse events) was a given to all three patients and resulted in a dramatic improve- constellation of symptoms consisting of severe burning and ment in the severity of this syndrome, with all of the events erythema of the face, chest, neck, ears, palms, soles, and geni- being reported as mild. talia (penis, testis, vagina, external labia). This reaction was In an attempt to additionally elucidate the nature of this often accompanied by conjunctival injection, itching of the mucocutaneous reaction, tryptase was measured in serum sam-

Fig. 1 Pharmacokinetic analysis of serum from patients treated with unlabeled c30.6 at doses of 10–50 mg/m2. The concentration of c30.6 in ng/ml (Y axis) is plotted against the time from infusion in days. An ELISA was used to determine the concentration of c30.6 in the serum (see “Patients and Methods”). Error bars are not shown for reasons of simplicity.

ples collected at baseline, 1-h, and 8-day time points from a measured in the first 28 days. For instance, the alkaline phos- number of patients (6, 8, 13, 15), including individual 13, who phatase fell by 50% in some individuals, whereas in others it had demonstrated the most florid reaction. None of these sam- increased to 30% above baseline. Similar changes were noted in ples demonstrated a significant rise in tryptase at the 1-h time ␥-glutamyltransferase and the transaminases. Overall there was point. Similarly, 1-h serum and 24-h urinary histamine and no significant change in liver function tests, which depended on methylhistamine levels were normal in a subset of three indi- patient dose or predicted response or was discordant with the viduals. pattern of their liver function tests in the subsequent 3 months. At the highest dose level, one individual (patient 17) ex- Pharmacokinetics. Pharmacokinetic analysis of plasma perienced mild abdominal pain and vomiting 1 h after the 123I-labeled c30.6 levels showed a biexponential clearance pattern commencement of the infusion, which lasted for 4 h. Analgesics with an ␣ half-life of 2.5 Ϯ 0.7 min (mean Ϯ SD) and a ␤-half-life were not required, because the symptoms were mild. The clin- of 46 Ϯ 15 h. An ELISA was used to determine the serum c30.6 ical examination was unremarkable, and serum amylase was 73 concentrations in those patients treated at the 10-, 25-, and 50- units/liter (normal Ͻ100 units/liter). The following day, this mg/m2 dose level (Fig. 1). At the 10 mg/m2 dose the ␤ half-life was patient developed an additional episode of mild abdominal pain 51 Ϯ 5 h, and this increased to 57 Ϯ 19 h at 25-mg/m2 dose level associated with two episodes of vomiting. On this occasion the and 81 Ϯ 15 h at 50 mg/m2. The maximum observed serum amylase was significantly elevated at 1030 units/liter, as was the concentration increased from 0.97 ␮g/ml at the 10-mg/m2 level to serum lipase (660 units/liter; normal Ͻ60 units/L), consistent 2.4 ␮g/ml at 25-mg/m2 and 7.9 ␮g/ml at 50-mg/m2 levels. Impor- with the diagnosis of acute pancreatitis. The symptoms rapidly tantly, at the 50-mg/m2 dose level, the serum concentration of c30.6 resolved without any treatment, and the amylase returned to remained at ϳ100 ng/ml from 1 week to at least 3 weeks after the normal within 48 h. Amylase levels on stored serum samples infusion. It is also of note that there was marked interpatient from six other patients (11–16) were found to be normal. Pan- variability in all of the pharmacokinetic parameters. creatitis was considered a dose-limiting toxicity, and therefore HACAs. Serum samples were assayed for HACAs at 2 the antibody dose was not escalated beyond 50 mg/m2. and 6 weeks as well as at 3 and 6 months. One of 17 patients All other adverse events were grade 1, except one instance of manifested a HACA response, and this was detectable at 14 days grade 2 tumor pain (sciatica) and one instance of rigors (patient 11). to 6 months posttreatment. This patient received only 2.1 mg The following events occurred up to 28 days after antibody admin- 123I-labeled c30.6 and was not treated with any unlabeled anti- istration but were not thought to be related to antibody administra- body. The level of HACAs was low (14 ng/ml at 3 and 6 tion: death from an acute myocardial infarct at 18 days and two months) and could be detected only in neat serum. In compar- instances of a dry throat and mouth secondary to promethazine. ison, sheep immunized with c30.6 have anti-c30.6 levels of The following events were probably related to antibody adminis- ϳ400 ␮g/ml in this assay. tration: gritty eyes for 4 days, vomiting and nausea for 2 days after Tumor Response. Whereas all 17 patients had measur- infusion (2 patients, grade 2), and anorexia (2 days). able disease, tumor response was only assessable in 13 of these Full Blood Count/Biochemistry/Liver Function Tests, individuals (Table 1). Two patients died before imaging could and Complement. No clinically significant changes in hema- be undertaken (one from a myocardial infarct and the second tology, biochemistry, or complement (C3, C4, CH50), occurred from progressive disease) and an additional two patients refused in the 4-week period after antibody administration. There was follow up scans (one withdrew from study and the other re- considerable interpatient variability in the liver function tests mained on study but did not undergo imaging). There were no

Fig. 2 PET fluorine-18-deoxyglucose (FDG) scanning of patient 13 after injection of 48 mg c30.6 antibody. Scans taken at baseline (A) and again at 7 days (B) and 6 weeks (C). The percent- ages of reduction in glucose uptake in (B) and (C) were 12–14% and 24–28%, respectively, com- pared with baseline.

partial or complete responses in the 10 assessable patients who ␮g/liter at 8 weeks (normal Ͻ3.0 ␮g/liter), yet CT scanning received unlabeled c30.6, with or without 123I-labeled c30.6. At clearly showed progression of disease. 4–6 weeks, five patients had progressive disease, and two had Biodistribution and Imaging. Gel filtration chromatogra- stable disease. The remaining three patients (8, 13, 15) had evi- phy was used to analyze serum collected at 4, 24, and 48 h from dence of improvement in at least some of their metastatic lesions. two patients who received only 123I-labeled c30.6. The radioactiv-

For instance, in patient 8 there was no change in the CT scan ity from all of the samples eluted as a single peak at a Mr of performed at 7 weeks; however, the PET scan demonstrated a 160,000, indicating that there was no detectable complexing or 22–30% reduction in glucose metabolism at measurable sites breakdown of the labeled antibody in vivo. Further evidence sup- within the extensive pelvic disease. The PET scan also showed porting the in vivo stability of the antibody and label was provided progressive disease in the chest, with an increase in the size and by analysis of 48-h urine collections, which showed that 17–21% avidity of lesions at the base of right lung, hilum, and mediastinum. of the injected activity was excreted in this period. This progression was not noted on CT but subsequently became Whole body planar images obtained at 0, 4, 24, and 48 h readily apparent. Figure 2 shows the results of PET scanning of after injection indicated that there was a very rapid and intense patient 13, where the qualitative decrease in glucose avidity of uptake of activity in the liver, coupled with a decrease in the some hepatic lesions is readily apparent at 7 days, with an addi- blood pool activity over the first hours (Fig. 3). At the zero time tional decrease at 6 weeks. The 6-week scan also identified new point, planar images of the four patients injected with 123I- lesions, particularly in the chest. These new lesions were also not labeled c30.6 (patient 1–4; Table 1) showed that 50% (range, detected on CT. Patient 15 refused CT scanning, but a PET scan 43–54%) of the dose localized in the liver, and at 48 h the performed at day 13 demonstrated a 7% decrease in glucose uptake localization remained high at 44% (range, 35–50%). However, in the extensive intra-abdominal disease. By 7 weeks, this patient as expected, the preinfusion of unlabeled c30.6 significantly had deteriorated clinically, and PET scanning confirmed progres- reduced the hepatic uptake of 123I-labeled c30.6 (mean hepatic sive disease, with a 24–35% increase in glucose uptake. localization, 25%; range, 21–35% at the zero time point). In The median percentage change in CEA from baseline to 8 patients 1–4 the mean whole-body effective dose equivalent weeks was found to be ϩ11% (range, Ϫ20 to ϩ169), 0% (range, was 3.19 Ϯ 0.37 ϫ 10Ϫ2 mGy/MBq. Ϫ38 to ϩ132), and ϩ6% (range, 0- ϩ54%) at the 10-, 25-, and SPECT obtained at both 24 and 48 h after 123I-labeled 50-mg/m2 dose levels. All patients in whom PET scanning c30.6 injection identified primary and some metastatic lesions. showed some reduction in particular sites of disease were also The two primary tumors (patients 7 and 11) that had not been found to have small increases in serum CEA levels, probably resected before study were successfully imaged as hot lesions reflecting the previously mentioned occurrence of progression at with radioactivities of 1.2 and 2% (Fig. 4A). Hepatic lesions other sites. Other patients, such as patient 10, demonstrated a were visualized either as hot lesions, cold lesions, or regions of sustained decrease in CEA from 195 ␮g/liter at baseline to 120 low activity surrounded by a hot rim (Fig. 4B). Although all of

Fig. 3 Whole-body planar images of patient 11 showing intense hepatic uptake of 123I-labeled c30.6.

low-energy ␥ emission (159 keV) allow for safe handling and improved imaging. This study demonstrated that 123I-labeled c30.6 antibody was of insufficient specificity or sensitivity for use as a diag- nostic imaging agent. Although some lesions such as primary colorectal tumors were readily detected, other large tumor deposits in the lungs and lymph nodes remained occult. The detection of hepatic lesions was clearly disadvantaged by the combination of the high uptake of antibody in the liver and the short half-life of the label. Previous studies have demonstrated that this type of imaging problem can be overcome by the use of antibody fragments and alternative labels (16–18). The whole- Fig. 4 SPECT images of a primary rectosigmoid cancer (A, arrow) and body planar images graphically demonstrated the intensive and a hot hepatic lesion (B, arrow). The images are coronal slices taken 24 h after the injection of 123I-labeled c30.6. prolonged uptake of antibody into the normal liver parenchyma. Furthermore, it was shown that this nonspecific uptake could be reduced by the preinfusion of cold antibody. It is likely that nonspecific hepatic uptake was one of the factors determining the individuals with hepatic disease were reported to have the antibody half-life and maximum serum concentrations. abnormal areas of hepatic uptake, SPECT did not identify all of These parameters increased as the dose of infused antibody was the lesions reported on CT and PET scanning. As expected, the escalated, probably as a result of saturation of hepatic binding pattern of uptake was somewhat dependent on the size of the sites. The reservoir of antibody in the liver and its slow release lesion. This observation is consistent with previous reports in back into the circulation may also account for the prolonged which lesions with a diameter of Ͼ2.5 cm usually appeared as maintenance of serum c30.6 levels in patients who received cold defects with or without a hot rim, whereas lesions Յ2.0 cm doses of 50 mg/m2. Despite the intensive hepatic uptake, there tended to show high uptake of radioactivity (15). Although two was no biochemical or clinical evidence of hepatotoxicity. patients with extra-abdominal disease (patients 2 and 13) par- The serum half-life and maximum serum concentration ticipated in the study, SPECT imaging did not identify the seen with c30.6 (81 h, 7.9 ␮g/ml at the 50-mg/m2 dose) is pulmonary and nodal tumor sites in either case. However, one comparable with that of other chimeric antibodies presently in individual with extensive pelvic disease did demonstrate a quite clinical trial. For instance the half-life of chimeric 17.1A was marked uptake of isotope at these sites (patient 8). 100 h, and the maximum serum concentration was 11 ␮g/ml when used at a doses of 40 mg fortnightly for three infusions DISCUSSION (19). Similarly, the IDEC-C2B8 had a half-life of 33 h after a In this study we describe the generation of a novel chimeric dose of 375 mg/m2, and this increased to 76 h after four antibody and its clinical evaluation in individuals with meta- infusions, as a result of saturation of CD20 sites in the serum static colorectal cancer. To obtain additional data on biodistri- and circulation (1). On the basis of these and other studies, we bution, pharmacokinetics, and tumor targeting, we also treated a anticipate that the half-life and maximum serum concentration subset of patients with radiolabeled c30.6 antibody. 123I was of c30.6 will increase after the infusion of multiple doses. chosen for this purpose, because its short half-life (13 h) and Most side effects occurred within 4 h of the antibody

infusion, were self-limiting, and did not require admission to tryptase levels have been clearly demonstrated in bronchoalveo- hospital. Some of these side effects such as rigors and headache lar and nasal lavage fluids from allergic individuals, despite the have been frequently observed with other antibody therapies (3, presence of unchanged normal serum levels (24–26). 20, 21). The gastrointestinal side effects and in particular the Finally, it is possible that the reaction represents direct episodes of nausea and vomiting were also predictable, because antibody cross-reactivity with the involved mucocutaneous tis- c30.6 extensively cross-reacts with the gastric mucosa. sues. The c30.6 antibody does show limited cross-reactivity One patient who received the highest dose of c30.6 developed with eccrine gland and duct epithelium, and its binding may an antibody-induced pancreatitis, the symptoms of which were have provided an indirect signal for mast cell degranulation. mild and self-limiting and were disproportionate to the degree of Whether the mechanism is a direct or indirect one, we postulate elevation of serum amylase that was observed. It is probable that that the antibody induces a significant cutaneous mast cell the pancreatitis resulted from binding of antibody to pancreatic degranulation, which is not associated with systemic effects. structures. This may have taken the form of direct cytolysis of The murine 30.6 was chimerized to reduce its immunogenic- acinar cells or obstruction secondary to cross-reactivity with ductal ity and to enhance its cytotoxic effects. In a previous clinical trial epithelium. Cross-reactivity against structures in the normal pan- of the murine antibody, all of the patients developed human anti- creas had previously been identified in immunohistochemical stud- mouse antibodies (12), whereas only a single patient in the current ies on postmortem tissues; however, precise localization of the 30.6 study developed a HACA response. Importantly, this response was antigen was not possible because of the presence of extensive of a low level and in fact occurred in a patient who received only autolysis, as is typical of this tissue. Although the serum amylase a low level of antibody. The lack of immunogenicity of c30.6 has was normal in six other individuals, it seems that infusion of allowed a multidose study to proceed, and early results indicate that antibodies, e.g., other drugs such as thiazide diuretics and azathio- no HACAs occurs after the administration of four doses.6 prine, can cause pancreatic injury (22). The c30.6 antibody may kill tumor cells in vivo by at least To our knowledge, the constellation of mucocutaneous symp- two possible mechanisms. In vitro data indicate that it is capable toms seen in this study has not been described with other mono- of lysing human colorectal cells in the presence of human clonal antibodies or indeed with any other form of drug therapy. mononuclear cells, by ADCC, although it does not lyse the same The temporal progression of burning cutaneous erythema involving cells in the presence of either rabbit or human complement (13). at first the face and then the chest, genitalia, palms, and soles, in Although the function the 30.6 antigen has not yet been defined, succession, was particularly distinctive. Pain at these sites was it is also possible that the binding of antibody could exert a often of such severity that it necessitated the use of narcotics. direct cellular effect, in a manner similar to the effects of Although there was some variability between individuals, it was antibodies, which bind to the HER-2/neu receptor (27, 28). apparent that the frequency and severity of this reaction increased Despite these potential actions, there were no partial responses proportionally to the dose of antibody administered. observed in this clinical trial. This finding is not dissimilar to Interestingly, this reaction occurred in the absence of symp- that of many other early studies of single doses of monoclonal toms classically associated with hypersensitivity reactions, such as antibodies in the treatment of solid tumors (29–31). bronchospasm, urticaria, facial and laryngeal edema, and hypoten- At present, antibody-based therapeutics represent an expen- sion. However, although the established reaction was refractory to sive treatment option. In initial phase studies of new antibodies, it all attempted therapies, it was readily prevented by premedication is therefore important to identify those modes and time points for with high doses of combination H1 and H2 antagonists. The imaging studies that will allow the earliest detection of treatment importance of high doses of these antagonists was recently illus- responses. In this regard, an important observation from this study trated in a follow up multidose trial of c30.6, where a 25% reduc- was the potential role of PET scanning. Using this modality, we tion in the dose of the H1 antagonist was associated with recurrence found that three patients had some objective disease improvement, of this syndrome (unpublished observations). an event that was paralleled by symptomatic improvement in two A number of pathogenic mechanisms can be considered in of those cases. This suggests that PET scanning may prove more regard to this mucocutaneous reaction syndrome. The possibility sensitive than computerized tomography in the early detection of that the reaction was the result of infusion of histamine contam- treatment response, particularly in the setting of extensive or bulky inating the antibody preparations was excluded by retrospective disease. A current limitation of PET scanning is the difficulty in analysis of all of the batches of antibody (23). A more likely precisely quantifying responses. Nevertheless, it is likely that this explanation was that the syndrome was the result of mast cell modality, perhaps when used in conjunction with other imaging degranulation, either directly or indirectly mediated by antibody tools, will provide a useful means for the early identification of infusion. Mast cell degranulation causes the release of a range of responses to monoclonal antibody therapy. We are presently inves- vasoactive and neuroactive mediators, such as histamine, tigating this issue as part of a multidose study with 30.6. tryptase, and prostaglandins (24), all of which have pharmaco- In summary, the chimerized c30.6 antibody is not immu- logical actions consistent with the vasodilation, pain, and flush- nogenic in humans and appears worthy of further study. It does, ing of the skin seen after infusion. The protein tryptase is also however, produce a unique profile of side effects, which al- released during mast cell degranulation and serves as a useful though significant can be well controlled with appropriate pre- marker of this event because of its long serum half-life. How- medication. Undoubtedly, the outcome of the multidose trial and ever, serial estimations of tryptase and histamine in symptomatic individuals in this study failed to show elevation of serum levels. The possibility remains that mast cell degranulation may have occurred only at a local level. In this regard, elevated 6 Unpublished observations.

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Robyn L. Ward, Deborah Packham, Anne M. Smythe, et al.

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