Imaging Organ Function in Small Animals
Image Analysis for Quantitative Assessment of Tumor Response to Therapy
Radiolabeled Monoclonal Antibody Imaging of Tumors and Positron Emission Tomography Oncology
Chemical Modifications of Antibodies for Tumor Targeting
Gene-specific Radiotherapy
INTRAMURAL
RESEARCH PROJECT
Z01 CL-00401-12 NMIP
October 1, 1999 to September 30, 2000
Title of Project:
Imaging Organ Function in Small Animals
Principal Investigator:
M.V. Green (Senior Investigator)
IPL, NMD, CC, NIH
Bethesda, MD 20892
Other Personnel:
J. Seidel, Physicist, Staff Scientist, NM
J.J. Vaquero, Visiting Fellow, NM
I. Lee, Visiting Fellow, NM
Collaborating Units:
PI, NCRR (J. Sullivan, Instrument Maker)
PET, CC (W. Eckelman, Radiochemist)
Staff-Years:
3.5
Human Subjects:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: Investigations undertaken during the past several years identified optimal subsystem designs for a stationary ring, small animal positron emission tomographic (PET) scanner with depth-of-interaction capability. These designs were acted upon during this reporting period, and components for this system were fabricated and procured. All 18 LGSO/GSO "phoswich" detector modules that will surround the animal were assembled and tested. Two three-module sectors were mounted on the gantry ring in opposition to one another and connected to in-houseÐdesigned signal-conditioning PC boards containing the module HV supplies and other support electronics. Signal lines emanating from these boards were connected to the data acquisition system that will actually be used in the final device. Initial measurements made with this partial ring suggest that the final system will exhibit a coincidence sensitivity much greater than any existing small animal scanner. Progress was also made toward completing the mechanical assembly of the system. Motors, drivers, power supplies, and screw-driven slides that will wobble the gantry and move the animal bed in all three dimensions were procured and tested, and plans for the layout of these components were formalized. It is expected that physical assembly of the system will be completed by the end of this reporting period and that the first images will be obtained by year's end. In addition to these activities, efforts were begun to procure a small animal computer tomography (CT) scanner to use in conjunction with this PET scanner. CT images of an animal obtained immediately before or after a PET imaging session will be used to correct the PET images for radiation attenuation and other degrading effects and to aid in identification of radiolabeled structures.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-00416-02 NMIP
October 1, 1999 to September 30, 2000
Title of Project:
Image Analysis for Quantitative Assessment of Tumor Response to Therapy
Principal Investigator:
S.L. Bacharach, Ph.D.
DNM, CC, NIH
Bethesda, MD 20892
Other Personnel:
J. Carrasquillo, M.D., CC
S. Libutti, M.D., NCI
K. Kurdziel, M.D., DRRP Fellow
P. Mansour, Ph.D., CC/NCI (NCI IRTA Fellow)
F. Jousse, M.D., CC (former Fogarty Fellow and Volunteer)
P. Choyke, M.D., DRD
Collaborating Unit:
DRD, NCI
Staff-Years:
3.5
Human Subjects:
x (a) Human subjects (b) Human tissues (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: The Department of Nuclear Medicine, in conjunction with the National Cancer Institute and the Department of Radiology, performs clinical research in the use of imaging in oncology. In particular, they are studying the use of positron emission tomographic (PET) images, in conjunction with computer tomography (CT) and magnetic resonance (MR) images, to evaluate the effects of therapy on tumors. Several therapeutic agents are being studied, among them various anti-angiogenesis therapies. The PET scanners are used to measure glucose metabolism, blood flow, and blood volume in tumors over the course of therapy. CT scans are used to determine tumor morphology, and MR imaging is used to determine both morphology and parameters related to tumor perfusion.
This research is geared toward developing, implementing, and testing methods to better quantify the data obtained from the images and to determine if these methods are efficacious for the monitoring of tumor therapy. These methods involve determination of tumor morphology and optimal determination of functional parameters such as blood flow, metabolism, and blood volume. The overall goal is the development of a clinically useful methodology for determining tumor response to therapy at an earlier phase of therapy than is currently possible. Such a methodology could permit optimal adjustment of the course of therapy while the therapy was still proceeding, potentially improving both tumor response and patient morbidity. Several areas of investigation are being pursued to achieve this goal, including these:
INTRAMURAL
RESEARCH PROJECT
Z01 CL-00600-07 NMRR
October 1, 1999 to
September 30, 2000
Title of Project:
Radiolabeled Monoclonal Antibody Imaging of Tumors and Positron Emission Tomography
Oncology
Principal Investigator:
J.A. Carrasquillo, M.D. (Deputy Chief, Senior Investigator)
NM, CC, NIH
Bethesda, MD 20892
Other Personnel:
C. Paik, Ph.D., Chemist, NM
M. Whatley, B.S., Technologist, NM
L. Park, Pharmacist, NM
D. Drumm, Technologist, NM
K. Wong, Chemist, NCI
Collaborating Units:
Metabolism Branch, DSC, NCI
Laboratory of Molecular Biology, DBS, NCI
Treatment Surgery Branch, DSC, NCI
Staff-Years:
4.8
Human Subjects:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: These studies are designed to develop improved methods for detecting and treating malignancies. Our group performs preclinical evaluation of antibodies that appear to be promising after initial screening by various laboratories at the National Cancer Institute and develops these antibodies for clinical application. The clinical trials evaluating their pharmacokinetics and dosimetry are performed by our group.
A collaborative radioimmunotherapy trial with Dr. Waldmann (PI), in which we used humanized anti-tac monoclonal antibody, is ongoing.
Various protocols using [F-18] FDG in PET and [O-15] water for tumor detection, followup, and blood flow measurements are ongoing.
We have begun preclinical studies evaluating pretargeting of antibodies for tumor therapy and have demonstrated therapeutic responses with Y-90 and Bi-213.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-02001-07 NMRR
October 1, 1999 to
September 30, 2000
Title of Project:
Chemical Modifications of Antibodies for Tumor Targeting
Principal Investigator:
J.A. Carrasquillo, M.D. (Senior Investigator)
NM, CC, NIH
Bethesda, MD 20892
Other Personnel:
C.H. Paik, Ph.D., Radiopharmaceutical Chemist
C.W. Park, Ph.D., NM
C.-H. K. Kao, Ph.D.
Z. Yao, M.D., Ph.D., NM
H.-J. Jeong, Ph.D.
Collaborating Units:
Metabolism Branch, DCS, NCI
Laboratory of Molecular Biology, DBS, NCI
Laboratory of Biochemistry, DBS, NCI
Staff-Years:
3.0
Human Subjects:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: This project was developed and is directed by C.H. Paik, Ph.D. The research has centered on improving the tumor-targeting property of monoclonal antibodies and the fragments by chemical modifications. For FY 2000 we have investigated the use of a novel multistep tumor targeting. Our strategy involves pretargeting tumors with monoclonal antibody (MoAb) peptide followed by the injection of a radiolabeled second peptide. This approach decouples the antibody injection from the radiolabel injection, thereby offering the specificity of antibody binding to tumor antigens while eliminating problems associated with slow blood clearance of radiolabeled MoAb due to its large size. This project involves the synthesis of MoAb-peptide conjugates, clearing agents, and radiolabeled peptides. We have been optimizing the synthesis of these reagents to maintain the integrity of the immunoreactivity of MoAb and allow dimerization formation of peptides. Our preliminary multistep tumor-targeting experiments using tumor-bearing nude mice showed high accumulation of a radiolabeled peptide in tumor tissues and low accumulation in non-tumor tissues and blood, thereby providing high tumor-to-background radioactivity ratios.
INTRAMURAL
RESEARCH PROJECT
Z01 CL-60001-05 NMRR
October 1, 1999 to
September 30, 2000
Title of Project:
Gene-specific Radiotherapy
Principal Investigator:
R.D. Neumann, M.D. (Chief, Senior Investigator)
NM, CC, NIH
Bethesda, MD 20892
Other Personnel:
I. Panyutin, Ph.D.
V. Karamychev, Ph.D.
E. Gaidamakova, Ph.D.
E. Chuang, B.S.
T. Winters, Ph.D.
I.V. Panyutin, M.D.
K. Mezhevaya, Ph.D.
E. Pastwa, Ph.D.
Collaborating Units:
NCI (V. Zhurkin, Ph.D.)
NIDDK (V. Malkov, Ph.D.; M. Levine, M.D.)
Epoch Pharmaceuticals (M. Reed, Ph.D.)
Somagenics Inc. (S. Kazakov, Ph.D.)
NIST (M. Dizdaroglu, Ph.D.)
Staff-Years:
6.5
Human Subjects:
(a) Human subjects (b) Human tissues x (c) Neither
(a1) Minors (a2) Interviews
Summary of Work: The goal of this project is the development of therapeutic radiopharmaceuticals based on targeting the decay of Auger-electron-emitting radioisotopes to specific sequences in DNA (genes) using triplex-forming oligonucleotides (TFOs) as delivery vehicles.
In in vitro studies we have demonstrated that TFOs are able to deliver Auger electron emitters to specific targets in cellular DNA in order to inactivate genes and/or kill the cells containing the target sequences. Decay of I-125 in TFOs results in strand breaks in both strands of the target DNA with an efficiency from 0.4-0.8 break/decay. Higher efficiency can be achieved with radionuclide multiple labeling. Breaks are confined to the triplex target sequence, and 90 percent of the sequence-specific breaks are located within 10 bp around the decay site. We showed that radiotoxicity of TFOs delivered into the cell nucleus as measured by clonogenic assay is 300 times less than that of DNA-incorporated I-125UdR.
TFOs were designed to target the human MDR1 gene that is amplified in KB-VI cells in culture. The TFOs were labeled with I-125, and the targeting was detected by the presence of radioiodine-induced breaks. The breaks were found in DNA purified from I-125-TFO-treated isolated nuclei and digitonin-permeabelized cells.
To increase the efficiency of targeting, a new generation of chemically modified oligonucleotides with increased in vivo stability permitting one-step labeling with Auger electron emitters is being developed. We have developed a rapid procedure for incorporation of the short life Auger electron emitters I-123 and I-111In-111 into ODNs and demonstrated that decay of these more clinically relevant radioisotopes produces DNA breaks with a yield comparable to that of I-125.
We also have shown that the fine structure of DNA damage by decay of Auger electron emitter depends on local DNA conformation and that by analyzing the DNA damage, one can obtain information on the structure of DNA in nucleoprotein complexes both in vitro and in vivo. Based on this principle, a new method of radioprobing DNA-protein complexes has been demonstrated in several model systems.
In addition, studies have been initiated to investigate the mechanisms of Auger-electronÐinduced DNA strand break repair in human cells. We have developed efficient methods of producing and isolating specific forms (form I and form II) of damaged shuttle vector plasmid DNA, using both oxidative agents and TFO-bound Auger-emitting radionuclides as damaging agents. A liposome delivery system has been developed for efficient delivery of damaged DNA into human cells in order to evaluate the in vivo repairability and mutagenicity of site-specific DNA double strand breaks (DSBs) induced by I-125-labeled TFOs. Using the methods described above, I-125-TFO-induced DNA DSBs were found to be very effective at inactivating a shuttle-vector-borne target reporter gene by mutagenic disruption. The mutation frequency for I-125-TFO-induced DSB was approximately 80 percent, and the mutation spectrum was dominated by multiple base deletions involving the targeted I-125 decay site. The I-125-TFO-induced DSB was also approximately 100 times more refractory to repair than oxidatively induced DSB similar to those produced by ionizing radiation and reactive oxygen species (ROS) such as hydroxyl radicals.
In vitro DSB repair assays have been developed to permit isolation of human proteins that are involved in DSB repair and to analyze DNA reaction products at the molecular level for comparison to DNA repaired in vivo. This assay employs plasmid DNA containing a DSB similar to that produced by ionizing radiation and other ROS. This DSB lesion more closely models naturally occurring DSB than DSB produced by other methods, such as restriction enzymes. In support of this assay, methods have been developed to produce and recover the large quantities of plasmid substrate DNA (linearized by bleomycin) necessary for chromatography and biochemistry procedures. The assay has been optimized for DSB rejoining using human HeLa cell extracts. Optimal conditions depend on the complexity of DSB introduced into the substrate DNA, with slight variations of pH and ionic strength being the variables. Standard reaction conditions have been established, and, under these conditions, the initial-repair-reaction rate for complex DSB produced by bleomycin is approximately twofold less than for the equivalent, but less chemically complex, restriction-enzyme-produced DSB.
The goals of the studies outlined above are to identify the human repair pathways involved in Auger-emitter-induced DSB repair; assess the consequences of repairing these lesions; and examine methods by which these repair processes can be manipulated to augment the radiotherapeutic effects of TFOs labeled with Auger-electron-emitting radionuclides.
Index:
Annual Report of Clinical Research Activities FY 2000
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