IJNM, 19(3): 53-67, 2004
Radioimmunotherapy (RIT) of Cancer Neeta Pandit-Taskar, Komal Jhaveri, Chaitanya Divgi Weill Medical College of Cornell University, New York, NY
T
he recent approval of two radiolabeled antibodies against CD20-positive lymphoma has led to a resurgence of interest in radioimmunotherapy. As with chemotherapy more than five decades ago, progress has been most marked in the hematologic neoplasms, both in myeloablative as well as non-myeloablative therapeutic strategies. Success in the radioimmunotherapy of solid tumors has been hampered by the immunogenicity of murine proteins and the relatively slow clearance of humanized intact immunoglobulins. Genetic engineering has enabled the development of a variety of antigen-binding constructs of various sizes and immunobiologic characteristics. Developments in radiochemistry as well as production of an increasing number of radionuclides with therapeutic potential and/or optimal imaging characteristics have spurred “tailored” therapeutic strategies which include dosimetry considerations of tumor burden. Such progress has generated pivotal studies that will establish the radiobiologic paradigms for successful radioimmunotherapy in solid tumor. This review will describe the current status of radioimmunotherapy in lymphoma and other hematologic neoplasms, and outline seminal studies that have paved the way to an understanding of radioimmunotherapy in solid tumors. Finally, the authors’ views of the future of this promising cancer therapy will be presented.
Introduction Radioimmunotherapy (RIT) is one among many therapeutic strategies that uses the “magic bullet” concept, whereby an antibody that recognizes tumor-associated antigens carries a cytotoxic radionuclide to target and destroy cancer cells. The initial studies with radiolabeled antibodies utilized polyclonal antibodies. These provided “proof of principle” that selective targeting to tumor was possible with immunoglobulins that recognized cancer-associated antigens. However, polyclonal antigens lacked uniformity and therefore were impractical to be developed as therapy. The discovery of hybridoma technology by Köhler and Milstein allowed the development of antibodies of uniform reactivity and immunobiologic characteristics. However, these monoclonal antibodies were produced in murine systems, and the invariable immunogenicity of murine proteins (in all but patients with BCorrespondence to: Chaitanya Divgi, MD Professor, Weill Medical College of Cornell University Attending Physician and Member, Memorial Sloan-Kettering Cancer Center 1275 York Avenue, New York, NY 10021. E-mail:
[email protected]
cell lymphoma) necessitated the development of less immunogenic chimeric and humanized antibodies. These chimeric and humanized monoclonal antibodies are now part of the therapeutic armamentarium of lymphoma and solid tumors; their slow serum clearance makes them ideal for immunotherapy but not suitable as carriers of cytotoxic radioactivity. RIT has the advantages of selective delivery of cytotoxic radiation following preferential uptake of radioantibody by tumor; high residence time in tumor; cross-fire effect by particle emissions and minimal deleterious effect on normal tissues. Radioimmunotherapy supplements the immune mechanisms of the unconjugated antibody therapy by its cross-fire and bystander effects and by overcoming the innate resistance of tumor cells to direct anti-tumor mechanisms. Selection of the optimal antigen-binding construct and radionuclide are important aspects in designing safe and effective therapy, especially for solid tumors. Impediments to successful targeting of all antigen sites include increases in interstitial pressure especially in bulky disease, changes in tumor microvasculature precluding access, and heterogeneous antigen distribution. Recombinant techniques have resulted in a variety of antigen-binding constructs of varying size, valency and immunobiologic characteristics selected to 53
Neeta Pandit et al address these problems. Moreover, radiobiologic principles can be applied toward rational design of agent, as advances in radiochemistry have made possible the stable conjugation of a variety of radionuclides to antigen-binding constructs. An understanding of radioimmunotherapy therefore requires an understanding of the potential and pitfalls of radionuclides in addition to an understanding of the promises and limitations of antigen-binding constructs.
Radionuclides with therapeutic potential Cytotoxic radionuclides can be broadly divided into four types: 1. Auger emitters and radionuclides that decay by internal conversion, including 125I and 67Ga; 2. Alpha emitters (213Bi, 211At); 3. Pure beta emitters, such as 67Cu and 90Y; and 4. Beta emitters that emit gamma radiation (131I, 177Lu, 186 Re, 166Ho). Auger emitters deposit high energy (Linear energy transfer) over extremely short distances and are therefore most effective when the decay occurs in the nucleus, and less so when the decay occurs in the cytoplasm. 125I is the prototypical radionuclide but with its long T1/2, it is less than optimal for therapy. Other similar radionuclides that have been studied, although not with antibodies, have included 111In. In both cases, the amount of radioactivity necessary is economically prohibitive. A radionuclide that is gaining increasing attention Table-1: Tradionuclides used for therapy with their halflives, energy and range in tissue (http://ie.lbl.gov/toi/nucSearch.asp). Radionuclide
T 1/2
Energy
131
I*#
8 days
β ave 191 Kev
90
#
2.67 days
β ave 934 Kev
Y
177
Lu*
6.7 day
β ave 150 Kev
186
Re*
3.78 day
β ave 362 Kev
188
Re*
17 h
β ave 795 Kev
166
Ho
26.8 h
β
62 h
β ave 141 KeV
At
7h
7.5 Mev (211 Po)
213
Bi*
46 min
8.4 Mev (213 Po)
225
Ac
10days
8.4 Mev
67
Cu#
211
* # 54
Photon _ β isotope
ave
666 Mev
in this category is 67Ga – though no clinical studies have been initiated yet, improvements in chelation chemistry have resulted in stable radioimmunoconjugates with 67Ga, and clinical trials are planned. Alpha emitters also deposit high energy, albeit over larger distances and therefore are extremely cytotoxic for small clusters of tumor cells (< 200µ). Antibodies conjugated with alpha emitters have shown promise in leukemia and when instilled into the surgically created brain tumor resection cavity, and will be most useful when there is rapid targeting of radioimmunoconjugate to tumor cells. However, this high linear energy transfer may preclude their use in solid tumor therapy (except perhaps in the adjuvant situation), where irradiation of normal tissue may preclude delivery of adequate cytotoxic radiation to the tumour. Beta emitters have been most widely used in systemic RIT as most studies and applications have been in established disease, and these nuclides can deposit energy over several millimeters. The linear energy transfer of betaminus energy is low; the distance of deposition is a function of the energy, being far greater for energetic nuclides such as 188 Re, than for 131 I. The first FDA-approved radioimmunotherapeutic agent consists of a pure (i.e. no gamma or other particulate emission) beta-minus emitting radiometal, yttrium-90. The other FDA-approved agent consists of a less energetic radionuclide, 131I, which has a gamma emission that permits external detection. Cytotoxic radionuclides may also be grouped according to their chemical characteristics into: Halogens (e.g. Iodine, 211 At); Metals (e.g. 90Y, 67Cu, 213Bi); Radio-lanthanides (e.g. 166 Ho, 177Lu); and Transitional elements (e.g. 186Re, 188Re). This classification is useful in the consideration of suitable linkers that can conjugate the radionuclide in a stable fashion to the antigen-binding construct. 131 I labeled antibodies have been most extensively studied in the treatment of cancer. Most of the studies have utilized direct iodination methods, usually using Iodogen®, a simple process that yields a stable compound in-vivo. Radioiodine has several advantages. 131I has been studied for several decades in the treatment of thyroid disorders (it is worth remembering that the specialty of Nuclear Medicine began as a therapeutic specialty involving 131I), and its biodistribution is well understood. Accumulation of free radioiodine can be successfully and easily prevented by coadministration of stable iodide and other forms of thyroid blockade including potassium perchlorate and thyroid hormone. High specific activity radioiodinated antibody that is stable for extended periods, permitting centralized production and distribution of large amounts of 131I labeled antibody, is now possible. However, radioiodine is suboptimal with antibodies that internalize (via clathrin coated
Indian Journal of Nuclear Medicine, Vol. 19 , No.3, September 2004 pits) subsequent to interaction with the antigen. Internalization leads to dehalogenation of the antigen– antibody complex and a consequent decrease in the cytotoxic potential. Radio-metal-labeled antibodies are not as susceptible to intracellular degradation and are thus preferred in internalizing systems. The most extensively studied radionuclide in RIT, after 131I, has been 90Yttrium.The first radio immunotherapeutic agent approved by the Food and Drug Administration (ibritumomab tiuxetan, Zevalin®), for CD20-positive B-cell lymphoma therapy, is a 90Y-labeled IgG (www.zevalin.com). 90Y is a pure beta-emitter, and the consequent inability to detect it by external devices has necessitated dosing schemes based on body weight or surface area. 111In has been used as a surrogate for imaging of 90Y; however its chemical characteristics are not identical; a true surrogate, the positron-emitting 86Y, has been used in other applications and is now being studied with antigenbinding constructs. The stable conjugation of 90Y to antibody is critical as free yttrium accumulates in bone. Conjugation is usually accomplished by a chelate; either free or chelated yttrium can dissociate from antibody – the latter is quickly cleared by glomerular filtration while the latter will accumulate in bone. Radio-metals such as 177Lu and 67Cu do not concentrate in bone, however, and are being explored – 177Lu has a gamma emission permitting external detection, while 64 Cu is a positron-emitting surrogate for the latter. The rhenium radioisotopes, transitional elements, are also being explored for their potential as cytotoxic agents. Both 186 Re and 188 Re have been used; 186 Re has been more extensively studied with intact IgG, though developments in conjugation chemistry have led to the study of short-lived 188 Re labeled to antibody fragments, particularly single chain (see below). The rhenium isotopes are gamma emitters and are eminently suitable for imaging with standard gamma cameras. 188Re has a shorter half-life and a more energetic beta-minus emission. The production of 188Re using a tungsten-188 generator system offers the potential of easy availability, while its relatively short (17 hour) half-life is likely to limit its use to loco-regional application or its conjugation with antibody fragments or smaller molecules. Recent developments in radiochemistry have made possible the study of alpha-emitters labeled to antibodies.Astatine-211 is a halide that has been stably conjugated to antibodies using a linker. Antibodies labeled with 211 At are being studied in loco-regional radioimmunotherapy trials in patients with malignant intracranial neoplasms. Bismuth-213 is being studied in myelogenous leukemias, and this radio-metal may be conjugated to antibodies using methodology established for other radio-metals. Its parent, actinium-225, emits a cascade of alpha particles and is being explored as in vivo “nanogenerator”.
Current Clinical Status of Radioimmunotherapy RIT in hematological malignancies Non – Hodgkin’s Lymphoma (NHL) is the fifth most common malignancy in the United States. An estimated 53,400 new cases of non-Hodgkin’s B-cell lymphoma are expected in the year 2003, with 23,400 deaths (1). NHL has the second fastest growing cancer death rate in the US (1). The most common forms of NHL are large B cell lymphoma and follicular lymphoma. Over 90% of B-cell lymphomas are CD20+ (2). NCCN Stage III or IV Indolent and low grade lymphomas such as follicular NHL are initially treated with immunotherapy and chemotherapy (anti-CD20 chimeric antibody rituximab with combination chemotherapy) (3). In the USA, radioimmunotherapy is currently approved for follicular lowgrade or transformed lymphomas that are refractory to rituximab. Although the RIT is with anti-CD20 antibodies, the added cross-fire effect of ionizing radiation results in increased efficacy in these situations. Other lymphoma antigens have also been targeted, including CD19, CD21b, CD22, and HLADR. Antibodies against CD22 have been studied extensively; the relatively slow clearance of humanized anti-CD22 antibody makes it an unlikely candidate for radioimmunotherapy (4, 5) Rituximab, directed against the B lymphocyte–specific antigen CD20, has demonstrated significant single-agent activity in follicular lymphoma, with response rates of 50% to 70% and median response duration of approximately 12 months (6-9). When rituximab is combined with chemotherapy, response rates in indolent lymphoma of 93% to 95% are frequently obtained (10, 11). A survival benefit has not yet been demonstrated in indolent lymphomas; there is thus a continuing need for new and effective agents to treat indolent lymphomas that are refractory to or have relapsed standard therapy. Two radiolabeled anti-CD20 antibodies – Ibritumomab tiuxetan (Zevalin®; IDEC Pharmaceuticals, San Diego, CA), a 90 yttrium-labeled agent, and tositumomab (Bexxar®, Corixa, Seattle, WA) a 131iodine-labeled agent - have been approved by the US Food and Drug Administration. Both 90Yibritumomab tiuxetan and 131I tositumomab were approved for use in patients with relapsed or refractory low-grade, follicular, or transformed B-cell NHL, including follicular lymphoma refractory to rituximab. These will be detailed. Ibritumomab Tiuxetan Ibritumomab, a murine IgG1 monoclonal antibody, is the parent murine antibody of the chimeric murine–human monoclonal antibody rituximab (12). The linker-chelator tiuxetan (MX-DTPA) is bound via a covalent bond to ibritumomab. Tiuxetan provides a high-affinity chelation site for the radioisotopes indium-111 or yttrium-90.The use of a murine antibody limits total-body irradiation due to the more 55
Neeta Pandit et al rapid clearance of murine (compared to chimeric) antibody. The therapeutic activity of the agent appears to be a combination of apoptosis induction, antibody-dependent cellular toxicity, and complement mediated cell lysis mediated by rituximab (13, 14) along with radiolysis caused by 90Y. A study (15) analyzing the data from a total of 179 patients in four clinical trials demonstrated the median radiation absorbed doses for 90Y (administered at 0.4 mCi/kg, up to a maximum of 32 mCi) were 7.42 Gy to spleen, 4.50 Gy to liver, 2.11 Gy to lung, 0.23 Gy to kidney and 0.57 Gy to total body. The median effective blood half-life was 27 h. Administration of the cold antibody before radioimmunotherapy diminishes nonspecific binding to circulating CD20+ B cells, improving biodistribution and tumor targeting, and theoretically saturates peripheral CD20 sites on bulky tumors, allowing greater penetration of the radioimmunoconjugate dose. The entire therapy can be carried out as an outpatient procedure (patient condition permitting), as 90Y is a pure beta-emitter. Radioimmunotherapy with ibritumomab tiuxetan is carried out in two phases, an imaging phase and a therapeutic phase. The first phase consists of an infusion of rituximab (250 mg/m2 administered following the same guidelines as for rituximab given alone) followed by an imaging dose of 5 mCi of 111Inlabeled ibritumomab tiuxetan. Whole-body gamma camera imaging is subsequently performed at selected time points (typically 2–24 hours after infusion and again 48–72 hours after infusion) to ensure acceptable biodistribution of the radioimmunoconjugate (a third image, obtained 90–120 hours after infusion, can be performed if necessary). Features of acceptable biodistribution include presence of radioactivity in the vasculature in early images with evidence of washout in the later images; increasing though not intense activity in the liver and spleen; and no evidence of increased radioactivity in the kidneys or the bowel. (These would all indicate the presence of an immune response to the murine protein.) Visualization of targeting to tumor is not a prerequisite for therapy. Dosimetry is not required before therapy in the defined patient population; imaging is carried out to exclude altered biodistribution that may predict increased toxicity (16). Figure 1 depicts anterior whole body planar images of a patient 2 & 48 hours after 111In-ibritumomab tiuxetan. Targeting to tumor is best visualized in the later images, while clearance from the blood pool and accumulation in the liver and spleen, though not in kidneys or bowel, is evident. The therapy phase, a week later, consists of another infusion of 250 mg/m2 rituximab followed by a therapeutic dose of 90Ylabeled ibritumomab tiuxetan, 0.3 to 0.4 mCi/ kg, up to a maximum dose of 32 mCi. (The dose is calculated based on the platelet counts: 0.4 mCi/Kg if the platelet count is > 150,000/ìL; 0.3 mCi/ Kg when the platelet count is between 100,000 and 149,000/ìL, up to a maximum in either case of 32 mCi; patients with platelet counts 15% normal marrow cellularity. It is also important to remember that although iliac crest biopsy is representative of overall marrow involvement, it is by no means comprehensive. Figure 2A is an example of a patient who had 15% iliac crest marrow involvement by lymphoma; the anterior whole body image clearly demonstrates involvement of the femoral shaft; the coronal SPECT slices (right) demonstrate targeting to known iliac and inguinal disease. This patient was treated to a whole body dose of 0.75 Gray, and suffered to unusual sideeffects. Individualizing treatment using dosimetric methodology can therefore be an important consideration in the choice of radioimmunotherapy. Dosimetry studies confirmed a 4-fold variation in the clearance rate (or effective half-life) of 131I-tositumomab (Figure 2B). Factors affecting clearance of the antibody include tumor size, splenomegaly, and the amount of bone marrow involvement. Due to variations in the clearance rate, the administered amount of radioactivity (in mCi) is adjusted individually to ensure that all patients receive the prescribed total body dose of 0.75 Gy. Using dosimetry with Iodine131-labeled antibodies enables physicians to directly measure the clearance rate in order to prospectively individualize the therapeutic dose. The treatment schema for the two agents remains similar (19). Patients receive a 450-mg infusion of unlabeled tositumomab (typically given over 1 hour) followed by 5 mCi of 131I-labeled tositumomab for dosimetry. Serial quantitative imaging (carried out after administration and before voiding; between 2 and 4 days; and finally between 6 and 7 days after administration) allows measurement of residence time of radioactive antibody, permitting calculation of radiation absorbed dose to the body based on patient mass using a simple look-up table (20). The therapeutic dose is administered a week later. Again, 450 mg of cold tositumomab antibody is followed by 131I-tositumomab, the amount of 131I now determined by the patient-specific dosimetry. Patients with platelet counts of >150,000/ìL are treated to a whole body dose no greater than 0.75 Gy; patients with platelet counts between 100,000 and 149,000/ìL aer treated to 0.65 Gy whole body radiation absorbed dose. A multicenter phase II trial assessed the safety and efficacy of the non myeloablative dose of the tositumomab regimen specifically in relapsed or refractory, low-grade, and transformed NHL (21). This trial interestingly showed that the ORR and CR rates were similar in patients with low-grade and transformed NHL (60% and 50%, respectively). Phase III clinical trials of patients with refractory or transformed low-grade B-cell NHL showed improved complete response rates and median response durations compared to their last prior chemotherapy. 58
As with all radioimmunotherapy, the dose-limiting toxicity of tositumomab is mainly myelotoxicity. Infusion related adverse reactions are minimal compared with those seen with ibritumomab, and both agents show minimal non hematologic toxicities. Because of dehalogenation, thyroid blockage is required for therapy with 131 I-tositumomab. Subclinical hypothyroidism (rise in serum thyroid-stimulating hormone (TSH)) may occur in about 7-10% of patients. Clinical hypothyroidism has not been reported. The annualized incidence of myelodysplasia and leukemia appears to be no greater than in patients who have not received radioimmunotherapy. Whereas low-dose therapy offers palliation, high-dose therapy may provide prolonged relapse-free survival and the potential for permanent cure (22). The toxicity of highdose therapy is greater, with more incidence of hypothyroidism. Second organ dose-limiting toxicity is cardiopulmonary with the dose-limiting (pulmonary) radiation absorbed dose being 27 Gy. Alpha emitters have also been investigated for potential use in treatment of NHL. A preclinical study of rituximab labeled with 211astatine appears to show promise for clinical trials (23), and clinical studies with shorter-acting 213Bi are also being carried out. Radio immunotherapy in other hematological malignancies In addition to its potential in treating B- cell Lymphoma, RIT has also shown considerable promise in the therapy of refractory Hodgkin’s Lymphoma and leukemia. Phase 1 trials have been carried out using 111In and 90Y – labeled polyclonal antiferritin to treat patients with refractory Hodgkin’s Lymphoma. These seminal studies were the first to demonstrate that myelotoxicity was dose-limiting (24). We have carried out a series of systematic studies with an anti-CD 33 antibody M195, labeled with a variety of cytotoxic nuclides (131I, 90Y, 213Bi), and have shown, especially with humanized M195 (IgG1), significant remission rates (25) in myelogenous leukemias, particularly acute promyelocytic leukemia (26). Initial trials with 131I were followed by studies with radiometals, after evidence that there is efficient internalization of M195 into target cells in vivo. Humanized M195 has improved avidity compared to murine M195 in addition to the potential abrogation of a human anti-antibody response; moreover, unlike murine M195 (IgG2b), humanized M195 mediates antibody-dependent cellular cytotoxicity against leukemia targets (27). When labeled with the beta-emitters 131I and 90Y, HuM195 eliminated large leukemic burdens in patients (28, 29), but produced prolonged myelosuppression requiring hematopoietic stem cell transplantation at high doses. Alpha emitters can selectively kill individual cancer cells with far fewer decay events than beta particle-emitting isotopes (30).
Indian Journal of Nuclear Medicine, Vol. 19 , No. 3, September 2004 To enhance the potency of native HuM195 yet avoid the nonspecific cytotoxicity of beta-emitting constructs, the alpha-emitting isotope 213Bi was conjugated to HuM195. This proved to be safe and feasible and was the first proof of concept for targeted systemic alpha immunotherapy in humans (31). We are now carrying out a Phase 2 study to determine the efficacy (clinical and molecular) of 213BiCHX(A)DTPA-huM195 in patients with refractory CD33positive leukemias.
RIT in solid tumors In contrast to lymphoreticular malignancies, patients with solid tumors have responded relatively poorly to RIT. The efficacy of RIT in solid tumor is affected by numerous factors, such as vascular and tumor permeability, development of human antimouse antibodies, heterogeneity of antigenic expression, the intrinsic radiosensitivity of the targeted tumor (32, 33). The development of solid tumor RIT has been slow, and few agents are in Phase 2 efficacy studies. The logical progression of studies, especially in colon cancer, using a variety of radionuclides and a variety of antigen-binding constructs in both non-myeloablative and myeloablative treatment regimens, has now led us to the threshold of rational design of both the clinical trials and radiolabeled antigen-binding constructs. The maximum number of clinical RIT studies in solid tumor have been carried out in colon cancer. Secreted antigen systems e.g. carcinoembryonic antigen (CEA) and TAG-72 (sialyl Tn); cell surface antigens including 17-1A, Lewis-y, and A33, and stromal antigens notably fibroblast activationprotein-alpha, have been studied, both at this Center and elsewhere. In this section, we will highlight clinical trials carried out with antigen-binding constructs against antigens that are expressed in solid tumors, largely colon cancer. When cancers other than colon cancer have been studied, these will be referenced. CarcinoEmbryonic Antigen (CEA) Goldenberg et al pioneered the use of antibodies in the detection and therapy of cancer, and they began with a radioiodinated polyclonal anti-CEA antibody (34). They have subsequently carried out RIT studies with 131I-labeled murine and humanized anti-CEA antibodies (35-38). Recent work has focused on humanized intact IgG huMN-14 (39). Excellent reviews of this extensive body of work have been published by Dr. Goldenberg (40, 41). As with all studies with radiolabeled intact antibodies, dose-limiting toxicity was hematopoietic and dependent upon radionuclide; immunogenicity of the murine antibodies precluded repeat therapy; non-myeloablative radioimmunotherapy has not resulted in lasting major responses; there is anecdotal
evidence of an improvement in survival. This group has also studied RIT in medullary thyroid cancer, a tumor in which CEA epression is frequent (42). Monoclonal antibodies for the detection and treatment of cancer were pioneered by Jean-Pierre Mach and his group, who were the first to develop anti-CEA monoclonal antibodies (43). They subsequently extended their observations to human studies with radionuclides, and have generated an impressive body of work demonstrating the relative targeting and biodistribution characteristics of a host of antigen-binding constructs (44-48). These studies further underscored the need for increasing tumor:background ratios of radiolabeled antibody, especially with respect to residence time, and showed that, despite a variety of nuclides attached to a variety of antigen-binding constructs, non-myeloablative radioimmunotherapy with anti-CEA constructs was unlikely to be of significant therapeutic benefit. Raubitschek et al, at the City of Hope Medical Center decided to focus on the development of non-immunogenic antigen-binding constructs against CEA, and have carried out studies with a chimeric (murine Fv genetically grafted to a human Fc backbone and grown in a mammalian cell line) antibody. The initial dose-finding study established no mass depenedence upon tumor targeting (49) and no evidence of immunogenicity. Subsequent RIT studies with 90Y-labeled antibody (50) demonstrated that radionuclide toxicity was, again, nuclide-dependent. No responses were observed in this non-myeloablative trial, prompting a myeloablative trial (51), the final results of which are awaited. The observation that non-myeloablative RIT with intact IgG is unlikely to result in major responses has spurred the development of various antigen-binding constructs (52), and these are now being produced for clinical study. In England, Begent et al have also carried out studies using 131I labeled antigen-binding constructs (whole IgG; F (ab)’2 fragments; cross linked divalent {DFM} and trivalent {TFM} versions). 131I labeled antibody showed high level of concentrations in the tumor, though not adequate to result in responses (53). Antibody-Directed Enzyme Prodrug Therapy (ADEPT) is a promising technique, pioneered by Bagshawe et al (54), that uses an antibody to target an enzyme selectively to a tumor where it converts a relatively non-toxic prodrug to a potent cytotoxic drug. Since the chemical conjugation has shown limited efficiency in tumor targeting , a recombinant fusion protein composed of MFE-23, an anticarcinoembryonic antigen (CEA) single chain Fv (scFv) antibody, fused to the amino-terminus of the enzyme carboxypeptidase G2 (CPG2) has been constructed to achieve ADEPT in CEA-producing tumors. The catalytic activity was measured after intravenous injection and it was found that its efficacy was good and also that it showed excellent tumor concentration and tumor retention. (55) 59
Neeta Pandit et al Begent et al have continued their pioneering studies by developing a single chain Fv fragment of their anti-CEA antibody. This scFv construct is grown in E. coli and exhibits impressive targeting abilities (56). TAG-72 (sTn/Tn) MAb CC49 is a murine IgG1 antibody which reacts with an antigen (Tn/sialylTn) expressed on a tumor-associated mucin, TAG-72. CC49 IgG exhibits high reactivity against tumor cells in most adenocarcinomas from colorectum, ovary, breast, stomach, and pancreas, with very little reactivity against normal tissues (57). As with most other antibodies with broad tumor specificities, CC49 was first studied in colon cancer patients in a Phase 1 RIT study with 131I-CC49 (58) after an initial study comparing CC49 with its lower affinity counterpart B72.3 had shown that CC49 had better relative uptake in colorectal cancer (59). Again, there were no clinical responses, dose-limiting toxicity was hematopoietic and nuclide-dependent, and immunogenicity was invariable. An attempt to reduce immunogenicity by administration of an immunosuppressive agent, deoxyspergualine (DSG) was less successful than desired (60), and so humanization of the antibody was embarked upon. In order to ensure that clearance of the humanized antibody was as close as possible to that of the murine intact IgG, the CH2 domain of the chimeric antibody was deleted. Initial clinical trials (61) have shown that clearance and targeting characteristics are indeed comparable and that the novel protein is non-immunogenic. Retention of the CH3 domain retains the immunobiologic functions of the antibody, and therefore a Phase 1 trial to assess both the immunobiologic properties of CH2-deleted huCC49 and the safety of 131I-labeled antibody, is underway. The initial studies in colon cancer were followed by Phase 1 and Phase 2 studies in a variety of other solid tumors, notably breast and prostate cancers (62-66). Intraperitoneal RIT with 131 I-CC49 has also been carried out in ovarian cancer (67). In all these studies, there has been excellent tumor targeting but no significant clinical responses. The limitations in the clinical application of radiolabeled CC49 IgG are primarily caused by normal tissue toxicity, immunogenicity, and relatively poor penetration into tumor. Genetically engineered single- chain antibody fragments (scFvs) are one way to potentially overcome some of these limitations. The scFvs had accelerated clearance from the vasculature, excellent penetration into the tumor from the vasculature, reduced immunogenicity, and higher tumor: background ratios than corresponding IgG, F (ab’)2 or Fab’ fragments in animal models (68-71). Larson et al. (72) showed that CC49 scFv is safe, that tissue equilibration and clearance are fast, and that same-day imaging of the primary and metastatic tumors is feasible in patients with colorectal carcinoma. However, due to their small 60
size and monovalency, scFvs clear the body too rapidly to allow for sufficient tumor uptake and retention for therapeutic applications. Moreover, early scFvs were generated in bacterial systems and may not have been too stable in vivo. Other antigenic systems Humanized monoclonal antibody A33 (huA33) targets the glycoprotein antigen A33, which is expressed on 95% of colorectal cancers, and normal colonic epithelium. We carried out Phase I/II 131I-mAb A33 RIT in patients with colon cancer. Again, DLT was nuclide-dependent and the MTD was determined to be 75 mCi/m2 131I. Although the isotope showed variable uptake in the normal bowel, gastrointestinal symptoms were mild or absent. (73) We also investigated 125I-murine A33, as the radioiodinated antibody appears to not undergo catabolism following internalization (probably via the macropinosome). If antibodies internalize and transport low-energy electron emitting isotopes close to the tumor cell nucleus, an improved therapeutic advantage may be achieved (74). This was the principle behind the Phase 1 study with 125I-A33 (75). The study was designed to determine the maximum-tolerated dose (MTD) of 125I-mAb A33, its limiting organ toxicity, and the uptake and retention of radioactivity in tumor lesions. MTD was not reached with doses of up to 350 mCi/m2 of 125I-mAb A33. There was modest antitumor activity in these heavily pretreated patients with lack of toxicity at the doses studied. Additional, significant responses were observed in those patients treated with chemotherapy [carmustine [BCNU], vincristine, fluorouracil, and streptozocin [BOF-Strep]) after completion of the 125ImAb A33 study. Murine 17-1A IgG2a antibody, which reacts against the surface epithelial antigen KSA, has also been studied with different radionuclides. The internalizing properties of murine antibody 17-1A in human colon cancer cells make it attractive as a carrier for 125I. The antibody was chimerized and a pilot clinical trial of increasing doses of 125I-chimeric 17-1A in patients with metastatic colorectal cancer was conducted. It was shown that high-dose outpatient radioimmunotherapy with a 125I-labeled internalizing antibody could be achieved without significant patient toxicity or radiation hazard. (76) Our team, under the leadership of Lloyd Old, has attempted to identify targets not only on cancer cell surfaces but also on other components of the tumor, including stroma and vasculature. We developed a novel targeting approach to colon cancer therapy by developing an antibody, F19, against fibroblast activation protein-alpha (FAP-á), which is highly expressed by activated fibroblasts abundant in most solid tumors. The antibody recognizes FAP-á, abundantly expressed by reactive stromal fibroblasts of epithelial cancers, including more than 95% of primary and metastatic colorectal carcinomas, but nor by normal tissue.
Indian Journal of Nuclear Medicine, Vol. 19 , No. 3, September 2004 Studies were carried out to define the toxicity, imaging, and biodistribution characteristics of iodine 131I-mAb F19. Due to selective localization in the tumor, with minimal uptake in the normal tissues, lesions as small as 1 cm could be picked up in the scans (77) . The easy accessibility of the FAP-positive tumor stromal fibroblasts to circulating monoclonal antibodies led to the humanization of this antibody, now named sibrotuzumab (BIBH 1). Phase 1 mass dose-finding studies have been completed with this novel protein (78), and RIT studies are being carried out. Renal cell carcinoma (RCC) is the most common form of kidney cancer. The prognosis for patients with metastatic
RCC is poor with a five-year survival rate of less than 10%. There is an urgent need for an effective treatment for RCC. Antibody G250, developed by Oosterwijk et al, recognizes carbonic anhydrase-IX, expressed in all tissue that contain the von Hippel Lindau (VHL) gene. Virtually all clear cell renal cancers express the antigen, and normal tissue cross-reactivity is limited to biliary epithelium (79). We carried out Phase I and II clinical trials with 131-labeled murine G250 (80); results were comparable to RIT in other solid tumors – the MTD was 75-90 mCi/m2 of 131I, immunogenicity was invariable and there were no major responses. We then undertook Phase 1 studies with 131I-chimeric G250. In order
Figure 3: Anterior and posterior whole body images 3 days after 131I-chimeric G250. Note excellent targeting to known lesions in scalp, hilum and lung. The CT and SPECT co-registered images show uniform distribution throughout the left hilar mass in the same patient. 61
Neeta Pandit et al to evaluate the effect of dose fractionation upon safety and efficacy, we simultaneously started a single large-dose RIT study based on escalating amounts of 131 I (81) and a fractionated RIT study using
