Lawrence E. Williams

Tumor localization of anti-CEA single-chain Fvs: improved targeting by non-covalent dimers

BACKGROUND Genetic engineering can produce novel antibody fragments with improved properties for applications such as tumor targeting in vivo. OBJECTIVES To produce stable monomeric (27 kDa) and dimeric (55 kDa) forms of a single-chain Fv (scFv) from the anti-carcinoembryonic antigen (anti-CEA) antibody T84.66, and assess the targeting and biodistribution properties in an animal model. STUDY DESIGN ScFv were constructed with either a 28 or 14 amino acid connecting peptide and expressed by secretion from E. coli. Following affinity purification, proteins were characterized by gel electrophoresis and mass spectrometry. Binding properties were assessed by size exclusion HPLC after incubation with antigen, and affinities determined by surface plasmon resonance. The shorter linker favored formation of dimers (and higher multimers) which showed unusual stability. ScFv were radiolabeled with 125I for tumor targeting and biodistribution studies of monomeric or dimeric forms were conducted in athymic mice bearing LS174T human colorectal carcinoma xenografts. RESULTS 125I-scFv monomers and dimers targeted exhibited rapid clearance kinetics in tumor-bearing mice. Nevertheless, the anti-CEA scFvs targeted very well to xenografts, leading to high tumor: normal organ ratios (greater than 20:1 at 24 h) for both forms. Tumor localization of the non-covalent dimers was much higher than monomers, reaching 10-15% injected dose per gram at 1 h. CONCLUSION Non-covalent dimers of scFv (also known as diabodies) are stable, easy to produce and show excellent targeting as compared to monomeric scFv, probably due to increased mass and valency.

Recommendations of the American Association of Physicists in Medicine on dosimetry, imaging, and quality assurance procedures for 90Y microsphere brachytherapy in the treatment of hepatic malignancies.

Yttrium-90 microsphere brachytherapy of the liver exploits the distinctive features of the liver anatomy to treat liver malignancies with beta radiation and is gaining more wide spread clinical use. This report provides a general overview of microsphere liver brachytherapy and assists the treatment team in creating local treatment practices to provide safe and efficient patient treatment. Suggestions for future improvements are incorporated with the basic rationale for the therapy and currently used procedures. Imaging modalities utilized and their respective quality assurance are discussed. General as well as vendor specific delivery procedures are reviewed. The current dosimetry models are reviewed and suggestions for dosimetry advancement are made. Beta activity standards are reviewed and vendor implementation strategies are discussed. Radioactive material licensing and radiation safety are discussed given the unique requirements of microsphere brachytherapy. A general, team-based quality assurance program is reviewed to provide guidance for the creation of the local procedures. Finally, recommendations are given on how to deliver the current state of the art treatments and directions for future improvements in the therapy.

Radioiodinated versus Radiometal-Labeled Anti–Carcinoembryonic Antigen Single-Chain Fv-Fc Antibody Fragments: Optimal Pharmacokinetics for Therapy

Antibody fragments with optimized pharmacokinetic profiles hold potential for detection and therapy of tumor malignancies. We studied the behavior of three anti-carcinoembryonic antigen (CEA) single-chain Fv-Fc (scFv-Fc) variants (I253A, H310A, and H310A/H435Q; Kabat numbering system) that exhibited differential serum persistence. Biodistribution studies done on CEA-positive tumor xenografted mice revealed that the 111In-labeled I253A fragment with the slowest clearance kinetics (T1/2beta, 27.7 h) achieved the highest tumor uptake (44.6% ID/g at 24 h), whereas the radiometal-labeled H310A/H435Q fragment with the most rapid elimination (T1/2beta, 7.05 h) reached a maximum of 28.0% ID/g at 12 h postinjection. The H310A protein was characterized by both intermediate serum half-life and tumor uptake. The 111In-based biodistribution studies showed that all three fragments were eliminated primarily through the liver, and hepatic radiometal activity correlated with the rate of fragment clearance. The 111In-labeled H310A/H435Q protein exhibited the highest liver uptake (23.5% ID/g at 24 h). Metabolism of the 125I-labeled scFv-Fc proteins resulted in low normal organ activity. Finally, the 125I/111In biodistribution data allowed for dose estimations, which suggest the 131I-labeled scFv-Fc H310A/H435Q as a promising candidate for radioimmunotherapy.

Pilot Trial Evaluating an 123I-Labeled 80-Kilodalton Engineered Anticarcinoembryonic Antigen Antibody Fragment (cT84.66 Minibody) in Patients with Colorectal Cancer

Purpose: The chimeric T84.66 (cT84.66) minibody is a novel engineered antibody construct (VL-linker-VH-CH3; 80 kDa) that demonstrates bivalent and high affinity (4 × 1010 m−1) binding to carcinoembryonic antigen (CEA). The variable regions (VL and VH) assemble to form the antigen-combining sites, and the protein forms dimers through self-association of the CH3 domains. In animal models, the minibody demonstrated high tumor uptake, approaching that of some intact antibodies, substantially faster clearance than intact chimeric T84.66, and superior tumor-to-blood ratios compared with the cT84.66 F(ab′)2 fragment, making it attractive for further evaluation as an imaging and therapy agent. The purpose of this pilot clinical study was to determine whether 123I-cT84.66 minibody demonstrated tumor targeting and was well tolerated as well as to begin to evaluate its biodistribution, pharmacokinetics, and immunogenicity in patients with colorectal cancer. Experimental Design: Ten patients with biopsy-proven colorectal cancer (6 newly diagnosed, 1 pelvic recurrence, 3 limited metastatic disease) were entered on this study. Each received 5–10 mCi (1 mg) of 123I-labeled minibody i.v. followed by serial nuclear scans and blood and urine sampling over the next 48–72 h. Surgery was performed immediately after the last nuclear scan. Results: Tumor imaging was observed with 123I-labeled minibody in seven of the eight patients who did not receive neoadjuvant therapy before surgery. Two patients received neoadjuvant radiation and chemotherapy, which significantly reduced tumor size before surgery and minibody infusion. At surgery, no tumor was detected in one patient and only a 2-mm focus was seen in the second patient. 123I-labeled minibody tumor targeting was not seen in either of these pretreated patients. Mean serum residence time of the minibody was 29.8 h (range, 10.9–65.4 h). No drug-related adverse reactions were observed. All 10 patients were evaluated for immune responses to the minibody, with no significant responses observed. Conclusion: This pilot study represents one of the first clinical efforts to evaluate an engineered intermediate-molecular-mass radiolabeled antibody construct directed against CEA. cT84.66 minibody demonstrates tumor targeting to colorectal cancer and a faster clearance in comparison with intact antibodies, making it appropriate for further evaluation as an imaging and therapy agent. The mean residence time of the minibody in patients is longer than predicted from murine models. We therefore plan to further evaluate its biodistribution and pharmacokinetic properties with minibody labeled with a longer-lived radionuclide, such as 111In.

Successful imaging of human cancer with indium-111-labeled phospholipid vesicles

Twenty‐four patients with proven primary and/or metastatic cancer received single intravenous injections of phospholipid vesicles containing 0.5 mCi of Indium‐111. Gamma camera scintigraphy 1 to 72 hours later visualized tumors in 22 patients (92%), including carcinomas of breast, lung, colon, prostate, kidney, cervix, thyroid, and soft tissue sarcoma, lymphoma, and melanoma. Tumor sites that were identified included soft tissues, bone, lung, liver, lymph node, and spinal cord. There were only two false‐positive images in metastatic sites and four false‐negative images in metastatic sites. Overall sensitivity for tumors in 97 individual sites was 85%, whereas specificity was 96%. Unsuspected areas of malignancy were seen in the lumbar subdural space, pleura, liver, thyroid, and lung. Besides tumor accumulations, homogeneous uptake was observed in normal liver and spleen. Radiation doses to these two organs were 2.2 and 2.9 cGy/0.5 mCi In‐111, respectively. Whole body radiation dose was 0.3 cGy/0.5 mCi. The use of Indium‐111‐labeled vesicles permits a wide variety of human tumors in primary and metastatic sites to be imaged without toxicity and with radiation doses comparable to other radionuclide scanning techniques.

The effect of tumor CEA content and tumor size on tissue uptake of indium 111-labeled anti-CEA monoclonal antibody†

This study was undertaken to determine the effect of tumor size and tumor carcinoembryonic antigen (CEA) content on the uptake of indium 111 (111In)‐labeled anti‐CEA monoclonal antibody in nude mice bearing xenografts. The tumor cell lines were WiDr, SW403, and LS174T, human colon cancer derivatives. The murine breast carcinoma cell line EMT‐6 was used as a control. Tumor CEA levels (ng/g) of tumor standard error of the mean [SEM], measured by enzyme immunoassay (EIA) were: EMT‐6, 0; WiDr, 105 ± 5.7; LS174T, 2052 ± 198; SW403, 17,575 ± 1,785. The 111In‐labeled monoclonal antibody was injected intravenously into mice bearing a single tumor. At 48 hours postinjection, scintiscan was performed, and the mice were killed so that biodistribution studies could be performed. The uptake of the monoclonal antibody was expressed as percent injected counts per minute per gram of tissue ± SEM. The non‐CEA‐producing tumor, EMT‐6, showed the lowest tumor uptake (1.4 ± 0.3). WiDr, an intermediate CEA‐producing tumor, showed some tumor uptake (16.4 ± 1.5). The high CEA‐producing tumors, SW403 and LS174T, had high tumor uptake (29.5 ± 5.0 and 51.1 ± 6.1, respectively). Biodistribution and scintiscan quality were closely related. Although LS174T had the best tumor uptake, SW403 had the highest CEA tumor content, indicating tumor CEA content cannot entirely predict scintiscan and biodistribution results. Tumor‐to‐blood (T/B), tumor‐to‐liver (T/L), and liver‐to‐blood (L/B) ratios were calculated for each animal and compared with tumor size. It was found that T/L had a negative correlation with tumor size (r = −0.72) and L/B had a positive correlation with tumor size (r = 0.94). These ratios may be useful clinically to follow response to therapy.

Targeted radionuclide therapy.

Targeted radionuclide therapy (TRT) seeks molecular and functional targets within patient tumor sites. A number of agents have been constructed and labeled with beta, alpha, and Auger emitters. Radionuclide carriers spanning a broad range of sizes; e.g., antibodies, liposomes, and constructs such as nanoparticles have been used in these studies. Uptake, in percent-injected dose per gram of malignant tissue, is used to evaluate the specificity of the targeting vehicle. Lymphoma (B-cell) has been the primary clinical application. Extension to solid tumors will require raising the macroscopic absorbed dose by several-fold over values found in present technology. Methods that may effect such changes include multistep targeting, simultaneous chemotherapy, and external sequestration of the agent. Toxicity has primarily involved red marrow so that marrow replacement can also be used to enhance future TRT treatments. Correlation of toxicities and treatment efficiency has been limited by relatively poor absorbed dose estimates partly because of using standard (phantom) organ sizes. These associations will be improved in the future by obtaining patient-specific organ size and activity data with hybrid SPECT/CT and PET/CT scanners.

AAPM Task Group 108: PET and PET/CT Shielding Requirements

The shielding of positron emission tomography (PET) and PET/CT (computed tomography) facilities presents special challenges. The 0.511 MeV annihilation photons associated with positron decay are much higher energy than other diagnostic radiations. As a result, barrier shielding may be required in floors and ceilings as well as adjacent walls. Since the patient becomes the radioactive source after the radiopharmaceutical has been administered, one has to consider the entire time that the subject remains in the clinic. In this report we present methods for estimating the shielding requirements for PET and PET/CT facilities. Information about the physical properties of the most commonly used clinical PET radionuclides is summarized, although the report primarily refers to fluorine-18. Typical PET imaging protocols are reviewed and exposure rates from patients are estimated including self-attenuation by body tissues and physical decay of the radionuclide. Examples of barrier calculations are presented for controlled and noncontrolled areas. Shielding for adjacent rooms with scintillation cameras is also discussed. Tables and graphs of estimated transmission factors for lead, steel, and concrete at 0.511 MeV are also included. Meeting the regulatory limits for uncontrolled areas can be an expensive proposition. Careful planning with the equipment vendor, facility architect, and a qualified medical physicist is necessary to produce a cost effective design while maintaining radiation safety standards.

Impact of radiolabeled antibody imaging on management of colon cancer

One hundred patients with known or suspected colorectal cancer were studied by radioimmunoconjugate scintigraphy prior to operation. Study subjects received murine monoclonal anticarcinoembryonic antigen labeled with indium 111 (Indacea). Sensitivity of imaging was 76 percent for primary tumors, 44 percent for hepatic metastases, 38 percent for extrahepatic abdominal metastases, and 78 percent for extraabdominal metastases. Seventeen of 46 patients (37 percent) with known or suspected hepatic metastases and no evidence of extrahepatic disease by conventional imaging methods had extrahepatic metastases at exploratory surgery. Nine of the 17 patients had disease accurately predicted by the Indacea scanning. The management of each of these nine patients was, or could have been, modified by the scan findings and unnecessary surgery eliminated. A number of patients without post-operative disease had an unexplained increase in plasma carcinoembryonic antigen level due to production of human antimouse antibody. The addition of excess mouse immunoglobulin to the plasma prior to assay blocked this artifactual increase.

The effects of hyperthermia on tumor carcinoembryonic antigen expression

The effects of hyperthermia on carcinoembryonic antigen (CEA) expression were investigated. The human colon adenocarcinoma cell line, LS174T, was heated in vitro for 42 degrees C/1 hr, 43 degrees C/1 hr, or 45 degrees C/30 min. Carcinoembryonic antigen membrane expression was assayed by live cell radioimmunoassay 0-6 days after heating. A heat exposure of 45 degrees C/30 min resulted in an initial decrease in carcinoembryonic antigen membrane expression 1 day post-heating followed by a 2-3 fold increase which peaked 3 days post-heating. Carcinoembryonic antigen expression began returning to normal by the sixth day. Heat exposures of 42 degrees C/1 hr and 43 degrees C/1 hr also resulted in increased carcinoembryonic antigen expression but this increase was of lesser magnitude and of shorter duration. Carcinoembryonic antigen shed into the medium, as measured by enzyme immunoassay, also increased after heating in a temperature-dependent fashion. Flow cytometry analysis demonstrated that cells in all phases of the cell cycle expressed this increase. We conclude that hyperthermia results in significant changes in carcinoembryonic antigen membrane expression and shedding. The implications that these findings have with regards to clinical hyperthermia and radioimmunotherapy are discussed.