Mammalian expression and hollow fiber bioreactor production of recombinant anti-CEA diabody and minibody for clinical applications.

Genetically engineered radiolabeled antibody fragments have shown great promise for the radioimmunoscintigraphy of cancer. Retaining the exquisite specificity of monoclonal antibodies yet smaller in molecular size, antibody fragments display rapid tumor targeting and blood clearance, a more uniform distribution in the tumor, and present a lower potential to elicit an immune response. However, one of the factors that has limited clinical evaluation of these antibody-derived proteins has been the difficulty in expressing and purifying the quantities necessary for clinical trials. This study outlines the capability of mammalian expression for the production of recombinant antibody fragments intended for clinical use. Two anti-carcinoembryonic antigen antibody fragments, the T84.66/212 Flex minibody (scFv-C(H)3) and the T84.66 diabody (scFv dimer) have been previously expressed and have shown excellent radioimaging properties in tumor bearing animals. To proceed toward human studies, these high affinity recombinant fragments and a second minibody version, the T84.66/GS18 Flex minibody, were expressed using a high-level mammalian expression system. Production of all three antibody fragments in a small-scale hollow fiber bioreactor resulted in 137-307 mg of crude antibody harvest. A purification protocol that employed ceramic hydroxyapatite and anion exchange chromatography resulted in 50-150 mg of purified T84.66 diabody and T84.66 minibody. The development of this level of research grade material established conditions for clinical production as well as provided material to complete pre-clinical studies and undertake protein crystallization studies. Scale-up for clinical studies produced 3.4 g of the T84.66 minibody in the harvest. A portion of this material was purified yielding 180 mg of highly purified T84.66 minibody intended for pilot radioimmunoscintigraphy studies of carcinoembryonic antigen (CEA) positive disease.

Tumor targeting of radiometal labeled anti-CEA recombinant T84.66 diabody and t84.66 minibody: comparison to radioiodinated fragments.

Recombinant antibody fragments offer potential advantages over intact monoclonal antibodies in the radioimmunoscintigraphy (RIS) of solid tumors. Due to their smaller molecular size, antibody fragments have shown rapid tumor targeting and blood clearance, a more uniform tumor distribution and a lower potential to elicit a human immune response. Previously, we have expressed two genetically engineered antibody fragments, the T84.66 diabody (scFv dimer) and the T84.66 minibody (scFv-CH3 dimer), specific to carcinoembryonic antigen (CEA). When radioiodinated, both antibody fragments exhibited rapid tumor targeting and rapid blood clearance in xenografted mice. To extend and optimize their future clinical RIS utility with radiometals, these antibody fragments were conjugated with the macrocycle 1,4,7,10-tetraazacyclododecane N,N',N' ',N' "-tetraacetic acid (DOTA) and labeled with 111In. Tumor targeting and biodistribution studies were carried out in athymic mice xenografted with a human colorectal tumor cell line, LS174T. The [111In]T84.66 diabody (55 kDa) exhibited very rapid tumor targeting with 12.5 +/- 0.4% injected dose per gram (% ID g(-1) +/- standard error) at 2 h and reached a maximum of 13.3 +/- 0.9% ID g(-1) at 6 h. However, kidney uptake was observed to reached a peak of 183.5 +/- 21.0% ID g(-1) at 6 h, a result similar to that reported by others for other low molecular weight fragments labeled with radiometals. Preadministration of an oral dose of D-lysine resulted in a 59% lowering of the renal accumulation at 6 h, but was accompanied by a 31% reduction of tumor uptake to 9.2 +/- 1.2% ID g(-1). The second recombinant antibody fragment, the [111In]T84.66 minibody (80 kDa), displayed rapid tumor targeting of 14.2 +/- 6.1% ID g(-1) at 2 h, and reached a maximum activity of 24.5 +/- 6.1% ID g(-1) by 12 h. Renal uptake achieved a plateau of 12-13% ID g(-1) which cleared to 7.2% ID g(-1) at 72 h. However, hepatic uptake was elevated and reached a maximum of 26.0 +/- 1.0% ID g(-1) at 12 h in these xenograft-bearing mice. Experiments in nontumor bearing mice showed a reduction of hepatic activity at 12 h to 16.6 +/- 1.5% ID g(-1), indicative of an intrinsic hepatic accumulation of the [111In]DOTA-T84.66 minibody or metabolites. While the anti-CEA [111In]DOTA-T84.66 diabody and T84.66 minibody retain the rapid tumor targeting properties of the radioiodinated form, the normal organ accumulation (kidneys and liver, respectively) of the [111In]DOTA forms appeared problematic for RIS and RIT applications. Development of alternative blocking strategies or new metabolizable chelates are under investigation to enhance the utility of the radiometal form of these and other promising recombinant antibody fragments.

Metabolism and renal clearance of 111In-labeled DOTA-conjugated antibody fragments.

Radiometal-labeled antibody fragments are promising reagents for radioimmunotherapy due to their high tumor uptake and rapid pharmacokinetics, but their therapeutic potentials are limited by high uptake and retention in the kidney. Identification of metabolic products is a first step in designing rationale approaches to lower kidney uptake. Previous studies in rats have shown that 111In-labeled DTPA-conjugated antibody fragments (via lysine residues) were degraded to an DTPA-epsilon-amino-lysine derivative and retained in the lysosomal compartments of the liver and kidney [Rogers et al. (1995) Cancer Res. 55, 5714s-5720s]. To determine the metabolic profile of another widely used metal-chelate, [111In]DOTA conjugated to lysines in antibody fragments via active ester chemistry, we analyzed kidney homogenates from nude mice injected with an [111In]DOTA-Fab generated enzymatically from the anti-lymphoma intact antibody Rituxan. The major kidney metabolite was identified as [111In]DOTA-epsilon-amino-lysine by comparison to an authentic synthetic standard. This end product was also identified in the urine, along with relatively small amounts of [111In]DOTA-Fab. Since injection of [111In]DOTA-epsilon-amino-lysine into nude mice resulted in rapid clearance into the urine without kidney retention, it is likely that the renal retention observed was due to kidney uptake of [111In]DOTA-Fab, followed by lysosomal degradation to [111In]DOTA-epsilon-amino-lysine, which is only slowly cleared from this compartment. This observation is supported by autoradiographs of the kidney showing rapid localization of radioactivity into the distal regions of the kidney cortex. To extend this analysis to clinical trials, we have also analyzed urine taken from a patient injected with the intact antibody [111In]DOTA-cT84.66. In that example, we found that the major radioactive species was also [111In]DOTA-epsilon-amino-lysine.

Numerical selection of optimal tumor imaging agents with application to engineered antibodies.

Three analytic indicators were used to compare five members of a monoclonal antibody (Mab) family. The cognates consisted of the genetically engineered intact chimeric IgGI (cT84.66) and related engineered fragments [scFv, diabody, minibody, F(ab')2] reactive against the same epitope of carcinoembryonic antigen (CEA). All analyses were based on radioiodinated Mabs targeting to colorectal xenografts of LS174T tumors in nude mice. Affinity constants were evaluated initially. A second indicator was the imaging figure of merit (IFOM) which determines how rapidly a statistically significant tumor image can be acquired. Finally, deconvolution was used to determine tumor temporal response to an arterial bolus. This last analysis gave the possible tumor accumulation in the absence of normal tissue sequestration. Affinities were all in excess of 10(8) M-1 and were highest for the divalent Mabs. Using the IFOM criterion, an 131I label was best suited as a radiolabel for the intact (IgG) T84.66, while an 123I label indicated optimal imaging with either minibody or F(ab')2. Deconvolution analyses showed that divalent members behaved similarly while the univalent member (scFv) had a tumor residence time smaller by an order of magnitude. The diabody had the largest impulse response function, but renal uptake may limit its present usefulness.

Multimerization of a chimeric anti-CD20 single-chain Fv-Fc fusion protein is mediated through variable domain exchange.

A series of single-chain anti-CD20 antibodies was produced by fusing single-chain Fv (scFv) with human IgG1 hinge and Fc regions, designated scFv-Fc. The initial scFv-Fc construct was assembled using an 18 amino acid (aa) linker between the antibody light- and heavy-chain variable regions, with the Cys residue in the upper hinge region (Kabat 233) mutagenized to Ser. Anti-CD20 scFv-Fc retained specific binding to CD20-positive cells and was active in mediating complement-dependent cytolysis. Size-exclusion HPLC analysis revealed that the purified scFv-Fc included multimeric as well as monomeric components. Variant scFv-Fcs were constructed incorporating four different hinges between the scFv and Fc regions, or three different linkers in the scFv domain. All formed multimers, with the highest level of multimerization found in the scFv-Fc with the shortest linker (8 aa). Elimination of an unusual salt bridge between residues L38 and H89 in the V(L)-V(H) domain interface failed to reduce the formation of higher order forms. Structural analysis of the scFv-Fc constructed with 18 or 8 aa linkers by pepsin or papain cleavage suggested the proteins contained a form in which scFv units had cross-paired to form a 'diabody'. Thus, domain exchange or cross-pairing appears to be the basis of the observed multimerization.

A phase I radioimmunotherapy trial evaluating 90yttrium-labeled anti-carcinoembryonic antigen (CEA) chimeric T84.66 in patients with metastatic CEA-producing malignancies.

Chimeric T84.66 (cT84.66) is a genetically engineered human/murine chimeric IgG, with high affinity and specificity to carcinoembryonic antigen (CEA). The purpose of this Phase I dose escalation therapy trial was to evaluate the toxicities, biodistribution, pharmacokinetics, tumor targeting, immunogenicity, and organ and tumor absorbed dose estimates of cT84.66 labeled with 90Y. Patients with metastatic CEA-producing malignancies were first administered 5 mCi 111In-labeled DTPA-cT84.66 (5 mg), followed by administration of the therapy dose of 90Y-labeled DTPA-cT84.66 1 week later. The therapy infusion was immediately followed by a 72-h administration of DTPA at 250 mg/m2/24 h. Dose levels of administered activity ranged from 5 to 22 mCi/m2 with three to six patients per level. Serial nuclear scans, blood samples, and 24-h urine collections were performed out to 5 days after infusion. Human antichimeric antibody response was assayed out to 6 months. Patients were administered up to 3 cycles of therapy every 6 weeks. Radiation absorbed doses to organs were estimated using a five compartment model and MIRDOSE3. Twenty-two patients received at least one cycle of therapy, with one individual receiving two cycles and two receiving three cycles of therapy. All were heavily pretreated and had progressive disease prior to entry in this trial. Reversible leukopenia and thrombocytopenia were the primary dose-limiting toxicities observed. Maximum tolerated dose was reached at 22 mCi/ m2. In general, patients with liver metastases demonstrated more rapid blood clearance of the antibody. Thirteen patients developed an immune response to the antibody. Average radiation doses to marrow, liver, and whole body were 2.6, 29, and 1.9 cGy/mCi 90Y, respectively. Dose estimates to tumor ranged from 66 to 1670 cGy (8.7 to 52.2 cGy/mCi 90Y) for each cycle of therapy delivered. Although no major responses were observed, three patients demonstrated stable disease of 12-28 weeks duration and two demonstrated a mixed response. In addition, a 41-100% reduction in tumor size was observed with five tumor lesions. 90Y-labeled cT84.66 was well tolerated, with reversible thrombocytopenia and leukopenia being dose limiting. Patients with extensive hepatic involvement by tumor demonstrated unfavorable biodistribution for therapy with rapid blood clearance and poor tumor targeting. Average tumor doses when compared with red marrow doses indicated a favorable therapeutic ratio. Stable disease and mixed responses were observed in this heavily pretreated population with progressive disease. This trial represents an important step toward further improving the therapeutic potential of this agent through refinements in the characteristics of the antibody and the treatment strategies used. Future trials will focus on the use of peripheral stem cell support to allow for higher administered activities and the use of combined modality strategies with radiation-enhancing chemotherapy drugs. Further efforts to reduce immunogenicity through humanization of the antibody are also planned. Finally, novel engineered, lower molecular weight, faster clearing constructs derived from cT84.66 continue to be evaluated in preclinical models as potential agents for radioimmunotherapy.

High-resolution microPET imaging of carcinoembryonic antigen-positive xenografts by using a copper-64-labeled engineered antibody fragment.

Rapid imaging by antitumor antibodies has been limited by the prolonged targeting kinetics and clearance of labeled whole antibodies. Genetically engineered fragments with rapid access and high retention in tumor tissue combined with rapid blood clearance are suitable for labeling with short-lived radionuclides, including positron-emitting isotopes for positron-emission tomography (PET). An engineered fragment was developed from the high-affinity anticarcinoembryonic antigen (CEA) monoclonal antibody T84.66. This single-chain variable fragment (Fv)-C(H)3, or minibody, was produced as a bivalent 80 kDa dimer. The macrocyclic chelating agent 1,4,7, 10-tetraazacyclododecane-N,N',N", N"'-tetraacetic acid (DOTA) was conjugated to the anti-CEA minibody for labeling with copper-64, a positron-emitting radionuclide (t(1/2) = 12.7 h). In vivo distribution was evaluated in athymic mice bearing paired LS174T human colon carcinoma (CEA positive) and C6 rat glioma (CEA negative) xenografts. Five hours after injection with (64)Cu-DOTA-minibody, microPET imaging showed high uptake in CEA-positive tumor (17.9% injected dose per gram +/- 3.79) compared with control tumor (6.0% injected dose per gram +/- 1.0). In addition, significant uptake was seen in liver, with low uptake in other tissues. Average target/background ratios relative to neighboring tissue were 3-4:1. Engineered antibody fragments labeled with positron-emitting isotopes such as copper-64 provide a new class of agents for PET imaging of tumors.

Truncation of blood curves to enhance imaging and therapy with monoclonal antibodies.

Targeting of monoclonal antibody (Mab) to solid tumor sites is a function of the blood curve of activity versus time. It has been suggested that the blood curve be artificially reduced to approach zero so that the contrast between tumor and blood uptake is maximized. We analyzed tumor uptake as a function of the time tc of blood curve truncation. By using a convolution approach, we were able to find the optimal times for setting the blood curve to zero in either diagnostic or therapeutic animal examples. Two iodinated cT84.66 anti-CEA engineered fragments, diabody and minibody, were considered using previous data from nude mouse studies involving the LS174T colorectal tumor model. Figures of merit (FOMs) were used to compare ordinary and truncated blood curves and their associated tumor accumulations. Using a 1231 label, it was seen that the appropriate time for diagnostic truncation occurred when tumor uptake, as measured, was a maximum. The corresponding point for therapy (with 1311 as a label) was at infinite time. We also demonstrated that the use of traditional indices led to ambiguities in the choice of truncation times. The traditional therapy index, the ratio of the integral of the tumor uptake to the integral of the blood uptake, was found to be a numerical constant independent of tc. This ratio was proved to be the integral of the tumor impulse response function. Use of such convolution techniques to assess truncation of the perfused material is probably also applicable to multistep processes as well as to lesion targeting with other tumor-specific pharmaceuticals.

Biodistribution and radioimmunotherapy of human breast cancer xenografts with radiometal-labeled DOTA conjugated anti-HER2/neu antibody 4D5.

HER2/neu oncogene encodes a 185 kDa trans-membrane protein which is overexpressed in 20-30% of breast and ovarian cancers and portends a poor prognosis. We have studied the targeting and therapy of this oncoprotein with 4D5, a murine monoclonal antibody which recognizes a distinct epitope on the extracelluar domain of HER2/neu. We conjugated the antibody with an active ester of the macrocyclic chelating agent DOTA, radiolabeled the conjugate with either (111)In or (90)Y, and studied the antibody distribution and therapy, respectively, in athymic mice bearing xenografts of MCF7/HER2/neu, a human breast cancer cell line transfected with the HER2/neu oncogene. For the biodistribution of (111)In-labeled DOTA-4D5, a high specificity of tumor localization (30% ID/g) was seen with a tumor-to-blood ratio of greater than 2 at 48 h postinjection. Compared to a previously published study with (125)I-labeled 4D5 in beige nude mice bearing NIH3T3/HER2/neu xenografts [De Santes et al. (1992) Cancer Res. 52, 1916-1923], (111)In-labeled 4D5 antibody gave superior antibody uptake in tumor (30% ID/g vs 17% ID/g at 48h). In the therapy study, treatment of the nude mice bearing MCF7/HER2/neu xenografts with 100 microCi (3 microg) of (90)Y-labeled DOTA-4D5 caused a 3-fold reduction of tumor growth compared to untreated controls (injected with human serum albumin) in 40 days. Treatment of animals with 100 microCi of nonspecific antibody (90)Y-labeled DOTA-Leu16 (3 microg) had no tumor growth inhibition. Treatment with unlabeled DOTA-4D5 (3 microg) had a slight effect on tumor growth compared to untreated controls. When analyzed at the level of single animals, no effect was seen in seven of nine animals; however, in two of the animals, tumor growth inhibition was observed. Although a cold antibody therapeutic effect was unexpected at this dose level (3 microg), it may be possible that in some animals that 3 microg of antibody of (90)Y-labeled DOTA-4D5 augmented tumor growth reduction. To further explore the effects of cold antibody treatment alone, animals were treated with 100 or 400 microg of unlabeled 4D5 administered in two doses. These animals showed a 1.7-1.8-fold reduction in tumor growth over 28 days, a result less than that obtained with RIT only.

Evaluating changes in stable chromosomal translocation frequency in patients receiving radioimmunotherapy.

PURPOSE: The lack of any consistent correlation between radioimmunotherapy (RIT) dose and observed hematologic toxicity has made it difficult to validate RIT radiation dose estimates to marrow. Stable chromosomal translocations (SCT) which result after radiation exposure may be a biologic parameter that more closely correlates with RIT radiation dose. Increases in the frequency of SCT are observed after radiation exposure and are highly correlated with absorbed radiation dose. SCT are cumulative after multiple radiation doses and conserved through an extended number of cell divisions. The purpose of this study was to evaluate whether increases in SCT frequency were detectable in peripheral lymphocytes after RIT and whether the magnitude of these increases correlated with estimated radiation dose to marrow and whole body. METHODS AND MATERIALS: Patients entered in a Phase I dose escalation therapy trial each received 1-3 intravenous cycles of the radiolabeled anti- carcinoembryonic antigen (CEA) monoclonal antibody, 90Y-chimeric T84.66. Five mCi of 111In-chimeric T84.66 was co-administered for imaging and biodistribution purposes. Blood samples were collected immediately prior to the start of therapy and 5-6 weeks after each therapy cycle. Peripheral lymphocytes were harvested after 72 hours of phytohemagglutinin stimulation and metaphase spreads prepared. Spreads were then stained by fluorescence in situ hybridization (FISH) using commercially available chromosome paint probes to chromosomes 3 and 4. Approximately 1000 spreads were evaluated for each chromosome sample. Red marrow radiation doses were estimated using the AAPM algorithm and blood clearance curves. RESULTS: Eighteen patients were studied, each receiving at least one cycle of therapy ranging from 5-22 mCi/m2. Three patients received 2 cycles and two patients received 3 cycles of therapy. Cumulative estimated marrow doses ranged from 9.2 to 310 cGy. Increases in SCT frequencies were observed after each cycle for both chromosomes 3 and 4 in 16 of 18 patients and in at least one chromosome for the remaining 2 patients. Cumulative increases in SCT frequencies ranged from 0.001 to 0.046 with no major differences observed between chromosomes 3 and 4. A linear correlation between cumulative marrow dose and increases in SCT frequencies was observed for chromosome 3 (R2 = 0.63) and chromosome 4 (R2 = 0.80). A linear correlation was also observed between increases in SCT frequency and whole body radiation dose or administered activity (R2 = 0.67-0.89). There was less correlation between observed decrease in wbc or platelet counts and marrow dose, whole body dose, or administered activity (R2 = 0.28-0.43). CONCLUSIONS: Increases in SCT frequency were detectable in peripheral lymphocytes after low dose-rate RIT irradiation. A linear correlation was observed between increases in SCT and marrow dose, whole body dose, and administered activity. This correlation provides one of the strongest radiation dose-response and activity-response relationships observed with RIT. The detection of SCT may therefore have application as an in situ integrating biodosimeter after RIT. This biologic parameter should prove useful in comparing effects on marrow for different therapeutic radionuclides and in comparing effects of RIT and external beam radiation doses on a cGy per cGy basis. As a result, this should allow for a more direct comparison between different methods of irradiation and in further refinement of radioimmunotherapy dose estimates and dosimetry methodology.

A method for patient-specific absorbed dose estimation for internal beta emitters.

The purpose of the study was to determine a technique for estimating patient-specific absorbed radiation doses in radioimmunotherapy and other internal emitter therapies. Beta Radiation sources were considered, with 90Y being the radionuclide of primary interest. Organ uptake of activity was determined using a merged set of computed tomography and planar nuclear images. Estimation of local absorbed dose was accomplished using a voxel source kernel. Voxel size was 0.2 x 0.2 x 0.5 cm; dimensions were from the digital resolution of the nuclear and computed tomography data sets. Dose-volume histograms were also obtained due to the voxel nature of the estimations. Organ dose estimates were made for two patients receiving the chimeric anticarcinoembryonic antigen antibody cT84.66. Considerable variation was observed when comparing the voxel kernel results with medical internal radiation dosimetry values obtained via the MIRDOSE3 program. Primary uncertainty in the organ dose estimates was determined to be due to the variation in organ mass. By correcting the S values in that program by the organ mass ratio, we found generally good agreement between our method and MIRDOSE3. We conclude that patient-specific absorbed doses can be estimated for 90Y-labeled antibodies.

Initial clinical experience evaluating Yttrium-90-chimeric T84.66 anticarcinoembryonic antigen antibody and autologous hematopoietic stem cell support in patients with carcinoembryonic antigen-producing metastatic breast cancer.

cT84.66 is a human/murine IgG1 with high affinity and specificity for carcinoembryonic antigen (CEA). An earlier Phase I trial defined the maximum tolerated dose for 90Y-diethylenetriaminepentaacetic acid (DTPA)-cT84.66 at 22 mCi/m2. Dose-limiting toxicities were reversible leukopenia and thrombocytopenia. The purpose of this Phase I trial was to evaluate the feasibility and toxicities of administering higher activities of 90Y-DTPA-cT84.66 with stem cell support in patients with CEA-producing breast cancer. Patients with CEA-producing breast cancer refractory to standard therapies underwent peripheral stem cell collection followed by infusion of 111indium-DTPA-cT84.66. Those patients demonstrating tumor targeting received a single therapy dose of 90Y-DTPA-cT84.66, followed by Ca-DTPA infusion for 72 h posttherapy. Stem cells were reinfused following a divided schedule. To date, seven patients have been accrued to this trial. Each patient received an imaging dose of (111)In-cT84.66. Six patients demonstrated tumor imaging and received a single cycle of 90Y-cT84.66 at 15 mCi/m2 (three patients) and 22.5 mCi/m2 (three patients). One patient did not demonstrate tumor imaging and was not treated. At these administered activities, 90Y-cT84.66 was well tolerated. No dose-limiting toxicities have been observed. All patients demonstrated hematopoietic recovery after stem cell infusion. One patient demonstrated stable disease for 4 months; one patient had stable disease and reduction of bone pain for 3 months; and a third patient experienced >50% reduction of an ovarian metastasis, resolution of malignant pleural effusion, stable pleural metastases, and stable bone scan for 14 months. Preliminary results from this ongoing Phase I trial are promising and demonstrate the feasibility and potential for antitumor effects of stem cell supported 90Y-cT84.66 therapy in patients with CEA-producing breast cancers.

A radionuclide therapy treatment planning and dose estimation system.

UNLABELLED: An object-oriented software system is described for estimating internal emitter absorbed doses using a set of computer modules operating within a personal computer environment. The system is called the Radionuclide Treatment Planning and Absorbed Dose Estimation System (RTDS). It is intended for radioimmunotherapy applications, although other forms of internal emitter therapy may also be considered. METHODS: Four software modules interact through a database backend. Clinical, demographic and image data are directly entered into the database. Modules include those devoted to clinical imaging (nuclear, CT and MR), activity determination, organ compartmental modeling and absorbed dose estimation. RESULTS: Both standard phantom (Medical Internal Radiation Dose [MIRD]) and patient-specific absorbed doses are estimated. All modules interact with the database backend so that changes in one process do not influence other operations. Results of the modular operations are written to the database as computations are completed. Dose-volume histograms are an intrinsic part of the output for patient-specific absorbed dose estimates. A sample dose estimate for a potential 90Y monoclonal antibody is described. CONCLUSION: A four-module software system has been implemented to estimate MIRD phantom and patient-specific absorbed doses. Computations of the doses and their statistical distribution for a pure beta emitter such as 90Y take approximately 1 min on a 300 MHz personal computer.

Intrapatient consistency of imaging biodistributions and their application to predicting therapeutic doses in a phase I clinical study of 90Y-based radioimmunotherapy.

Intrapatient variation in the biodistribution of the chimeric monoclonal antibody cT84.66 was assessed in 19 patients having a variety of carcinoembryonic antigen (CEA) positive tumors. The two studies, including whole-body imaging and blood and urine specimen collections, were conducted within 14 days of each other using (111)In-cT84.66 at a fixed total protein dose of 5 mg per patient per study. An initial pretherapy infusion of (111)In-cT84.66 was administered followed by a therapy coinfusion of (111)In-ct84.66 and 90Y-cT84.66 A closed five-compartment model was used to integrate source organ activity curves as residence time inputs into the MIRDOSE3 program. Normal organ absorbed doses were estimated for 90Y-cT84.66, the corresponding radiotherapeutic agent. For the two (111)In-cT84.66 biodistributions, all data were modeled with a R2 value of between 0.72 and 1.00 with the exception of the urine data taken during therapy. This was due to the need of diethylenetriaminepentaacetic acid during the therapy phase because of the possibility that yttrium might escape from the chelator attached to the antibody. With the assurance that the biodistributions were reproducible, we were able to estimate the 90Y-cT84.66 absorbed doses on a per-patient basis. Concordance coefficients showing the agreement between the imaging and therapy phase dose estimates were between the 0.60 and 0.99 levels for liver, spleen, red marrow, total body, and other organ systems. Median results were: 27, 17, and 2.7 rad/mCi of 90Y-cT84.66 for liver, spleen, and red marrow, respectively. Because of decreases in platelets and white cells as the amount of 90Y was increased, dose-limiting toxicity was found at 22 mCi/m2. We conclude that patient biodistributions were consistent over time to 14 days so as to allow absorbed dose estimation in a radioimmunotherapy trial involving the cT84.66 anti-CEA antibody.

Dose escalation trial of indium-111-labeled anti-carcinoembryonic antigen chimeric monoclonal antibody (chimeric T84.66) in presurgical colorectal cancer patients.

UNLABELLED: Chimeric T84.66 (cT84.66) is a high-affinity (1.16x10(11) M(-1)) IgG1 monoclonal antibody against carcinoembryonic antigen (CEA). The purpose of this pilot trial was to evaluate the tumor-targeting properties, biodistribution, pharmacokinetics and immunogenicity of 111In-labeled cT84.66 as a function of administered antibody protein dose. METHODS: Patients with CEA-producing colorectal cancers with localized disease or limited metastatic disease who were scheduled to undergo definitive surgical resection were each administered a single intravenous dose of 5 mg of isothiocyanatobenzyl diethylenetriaminepentaacetic acid-cT84.66, labeled with 5 mCi of 111In. Before receiving the radiolabeled antibody, patients received unlabeled diethylenetriaminepentaacetic acid-cT84.66. The amount of unlabeled antibody was 0, 20 or 100 mg, with five patients at each level. Serial blood samples, 24-hr urine collections and nuclear images were collected until 7 days postinfusion. Human antichimeric antibody response was assessed up to 6 mo postinfusion. RESULTS: Imaging of at least one known tumor site was performed in all 15 patients. Fifty-two lesions were analyzed, with an imaging sensitivity rate of 50.0% and a positive predictive value of 76.9%. The antibody detected tumors that were not detected by conventional means in three patients, resulting in a modification of surgical management. Interpatient variations in serum clearance rates were observed and were secondary to differences in clearance and metabolic rates of antibody and antibody:antigen complexes by the liver. Antibody uptake in primary tumors, metastatic sites and regional metastatic lymph nodes ranged from 0.4% to 134% injected dose/kg, resulting in estimated 90Y-cT84.66 radiation doses ranging from 0.3 to 193 cGy/mCi. Thirteen patients were evaluated 1-6 mo after infusion for human antichimeric antibody, and none developed a response. No major differences in tumor imaging, tumor uptake, pharmacokinetics or organ biodistribution were observed with increasing protein doses, although a trend toward increasing blood uptake and decreasing liver uptake was observed with increasing protein dose. CONCLUSION: Chimeric T84.66 demonstrated tumor targeting comparable to other radiolabeled intact anti-CEA monoclonal antibodies. Its immunogenicity after single administration was lower than murine monoclonal antibodies. These properties make 111In-cT84.66, or a lower molecular weight derivative, attractive for further evaluation as an imaging agent. Yttrium-90 dosimetry estimates predict potentially cytotoxic radiation doses to select tumor sites, which makes 90Y-cT84.66 also appropriate for further evaluation in Phase I radioimmunotherapy trials. Although clinically important changes in biodistribution, pharmacokinetics and tumor targeting with increasing protein doses of 111In-cT84.66 were not demonstrated, the results do suggest that antibody clearance from the blood is driven by hepatic uptake and metabolism, with more rapid blood clearance seen in patients with liver metastases. These patients with rapid clearance and potentially unfavorable biodistribution for imaging and therapy may, therefore, be a more appropriate subset in which to evaluate the role of administering higher protein doses. This underscores the need to further identify, characterize and understand those factors that influence the biodistribution and clearance of radiolabeled anti-CEA antibodies, to allow for better selection of patients for therapy and rational planning of radioimmunotherapy.

Monte Carlo-assisted voxel source kernel method (MAVSK) for internal beta dosimetry.

A method is described for the determination of patient-specific organ beta doses given a known cumulated internal radioactivity distribution. A voxel source kernel for 90Y analogous to the point source function was simulated. Dose to each organ of interest could then be estimated by convolving the voxel source kernel with the patient's 3-D volume with known radioactivity assigned to each voxel. The dose calculation on eight organs took less than 1 min per patient using a Sun Sparc10 workstation.

Biodistribution of 111In- and 90Y-labeled DOTA and maleimidocysteineamido-DOTA conjugated to chimeric anticarcinoembryonic antigen antibody in xenograft-bearing nude mice: comparison of stable and chemically labile linker systems.

Biodistributions of two radiometal chelate conjugates of the human/murine chimeric anticarcinoembryonic antigen monoclonal antibody cT84.66 were obtained in nude mice bearing LS174T human colorectal carcinoma xenografts. Derivatives of the macrocyclic chelating agent 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) were covalently attached to the antibody by a stable amide linkage and by a maleimidocysteineamido side chain (MC-DOTA) that has been shown to be chemically labile at physiological temperature and pH. Biodistributions of both 111In and 90Y labels were obtained in these studies. At common biodistribution time points, it was found that the 111In label had greater uptake in the liver than 90Y for both conjugates. No significant differences were found with respect to bone uptake of 90Y using either chelate. Blood curves were generally lower at comparable time points for MC-DOTA, indicative of faster clearance as compared to DOTA. Tumor uptake was high for both conjugates (57-68% ID/g at 48 h), with a longer tumor residence time in the case of the DOTA conjugate, probably a result of its longer blood circulation times. We conclude that bone uptake of 90Y would be minimal if either DOTA or MC-DOTA were used as the bifunctional chelator. This would imply preference for these macrocyclic ligands if radiation doses to the bone marrow would be considered to be dominated by skeletal uptakes. Alternatively, if bone marrow radiation dose is dominated by circulating antibody, the chemically labile linker system employed by the MC-DOTA conjugate offers the advantage of enhanced blood clearance.

Estimating residence times and their associated errors in patient absorbed-dose calculation.

OBJECTIVE: An approach for estimating organ residence times (tau) and their errors in patient internal emitter radiation dosage calculations has been determined. METHODS: Using a modeling algorithm and its associated parameters, chimeric anti-CEA monoclonal antibody (cT84.66) patient organ uptake data and residence times of source organ activity were calculated. Through the covariance matrix of the model's parameters and subsequent Monte Carlo simulations, errors in organ residence time (gamma tau) also were estimated RESULTS: These relative tau errors were found to be model-dependent; increasing as the number of organs being simultaneously modeled in a set of two patients being considered for 90Y-cT84.66 radioimmunotherapy. CONCLUSION: Use of modeling and Monte Carlo methods provide a general, direct procedure for calculating the degree of accuracy of activity integrals and other mathematical functions of kinetic variables.

Clinical evaluation of indium-111-labeled chimeric anti-CEA monoclonal antibody.

UNLABELLED: Chimeric T84.66 (cT84.66) is a high-affinity (1.16 x 10[11] M[-1]) IgG1 monoclonal antibody (MAb) against carcinoembryonic antigen (CEA). This pilot trial evaluated the tumor-targeting properties, biodistribution, pharmacokinetics and immunogenicity of 111In-labeled cT84.66. METHODS: Patients with CEA-producing metastatic malignancies were administered a single intravenous dose of 5 mCi 111In-diethylenetriaminepentaacetic acid-cT84.66. Serial blood samples, 24-hr urine collections and nuclear images were collected up to 7 days postinfusion. Human antichimeric antibody response was assessed up to 6 mo postinfusion. RESULTS: Imaging of at least one known tumor site was observed in 14 of 15 (93%) patients. Seventy-four lesions were analyzed with an imaging sensitivity rate of 45.1% and a positive predictive value of 94.1%. In one patient, two additional bone metastases developed within 6 mo of antibody administration at sites initially felt to be falsely positive on scan. One patient developed a human antichimeric antibody response predominantly to the murine portion of the antibody. The antibody cleared serum with a median T(1/2alpha) of 6.53 hr and a T(1/2beta) of 90.87 hr. Interpatient variations in serum clearance rates were observed and were secondary to differences in clearance and metabolic rates of antibody-antigen complexes by the liver. One patient demonstrated very rapid clearance of antibody by the liver, which compromised antibody localization to the primary tumor. Antibody uptake in primary and metastatic tumors ranged from 0.5% to 10.5% injected dose/kg, resulting in estimated radiation doses ranging from 0.97 to 21.3 cGy/mCi 90Y. Antibody uptake in regional lymph nodes ranged from 1.3% to 377% injected dose/kg, resulting in estimated radiation doses ranging from 2.0 to 617 cGy/mCi 90Y. CONCLUSION: Chimeric T84.66 demonstrated tumor targeting that was comparable to that of other radiolabeled intact anti-CEA Mabs. Its immunogenicity after single administration was lower than murine Mabs. These properties make cT84.66 or a lower molecular weight derivative attractive for further evaluation as an imaging agent. These same properties also make it appropriate for future evaluation in Phase I therapy trials. Finally, a wide variation in the rate of antibody clearance was observed, with one patient demonstrating very slow clearance, resulting in the highest estimated marrow dose of the group, and one patient demonstrating unusually rapid clearance, resulting in poor antibody localization to tumor. Data from this study suggest that serum CEA levels, antibody-antigen complex clearance and, therefore, antibody clearance are influenced by both the production and clearance rates of CEA. This underscores the need to further identify, characterize and understand those factors that influence the biodistribution and clearance of radiolabeled anti-CEA antibodies to allow for better selection of patients for therapy and rational planning of radioimmunotherapy.

Pharmacokinetic modeling and absorbed dose estimation for chimeric anti-CEA antibody in humans.

UNLABELLED: The objective of this article was to model pharmacokinetic data from clinical diagnostic studies involving the 111In-labeled monoclonal antibody (MAb) chimeric T84.66, against carcinoembryonic antigen. Model-derived results based on the 111In-MAb blood, urine and digital imaging data were used to predict 90Y-MAb absorbed radiation doses and to guide treatment planning for future therapy trials. Fifteen patients with at least one carcinoembryonic antigen-positive lesion were evaluated. We report the kinetic parameter estimates and absorbed 111In-MAb dose and projected 90Y-MAb doses for each patient as well as describe our approach and rationale for modeling an extensive set of pharmacokinetic data. METHODS: The ADAPT II software package was used to create three- and five-compartment models of uptake against time in the patient population. The "best-fit" model was identified using ordinary least squares. Areas under the curve were calculated using the modeled curves and input into MIRDOSE3 to estimate absorbed radiation doses for each patient. RESULTS: A five-compartment model best described the liver, whole body, blood and urine data for a subcohort of nine patients with digital imaging data. A three-compartment model best described the blood and urine data for all 15 clinical patients accrued in the clinical trial. For the subcohort, the largest projected 90Y-MAb doses were delivered to the liver (mean, 24.78 rad/mCi; range, 15.02-37.07 rad/mCi), with red marrow estimates on the order of 3.32 rad/mCi (range, 1.24-5.55) of 90Y. Corresponding estimates for the 111In-MAb were 3.18 (range, 2.09-4.43) and 0.55 (range, 0.34-0.74), respectively. CONCLUSION: The three- and five-compartment models presented here were successfully used to represent the blood, urine and imaging data. This was evidenced by the small standard errors for the kinetic parameter estimates and R2 values close to 1. As planned future therapeutic trials will involve stem cell support to alleviate hematological toxicities, the development of an approach for estimating doses to other major organs is crucial.