Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 69
Filter
1.
PLoS One ; 7(4): e34404, 2012.
Article in English | MEDLINE | ID: mdl-22545083

ABSTRACT

Theranostic agents are critical for improving the diagnosis and treatment of non-Hodgkin Lymphoma (NHL). The peptidomimetic LLP2A is a novel peptide receptor radiotherapy candidate for treating NHL that expresses the activated α4ß1 integrin. Tumor-bearing dogs are an excellent model of human NHL with similar clinical characteristics, behavior, and compressed clinical course. Canine in vivo imaging studies will provide valuable biodistribution and affinity information that reflects a diverse clinical population of lymphoma. This may also help to determine potential dose-limiting radiotoxicity to organs in human clinical trials. To validate this construct in a naturally occurring model of NHL, we performed in-vivo molecular targeted imaging and biodistribution in 3 normal dogs and 5 NHL bearing dogs. (99m)Tc-LLP2A-HYNIC-PEG and (99m)Tc-LLP2A-HYNIC were successfully synthesized and had very good labeling efficiency and radiochemical purity. (99m)Tc-LLP2A-HYNIC and (99m)Tc-LLP2A-HYNIC-PEG had biodistribution in keeping with their molecular size, with (99m)Tc-LLP2A-HYNIC-PEG remaining longer in the circulation, having higher tissue uptake, and having more activity in the liver compared to (99m)Tc-LLP2A-HYNIC. (99m)Tc-LLP2A-HYNIC was mainly eliminated through the kidneys with some residual activity. Radioactivity was reduced to near-background levels at 6 hours after injection. In NHL dogs, tumor showed moderately increased activity over background, with tumor activity in B-cell lymphoma dogs decreasing after chemotherapy. This compound is promising in the development of targeted drug-delivery radiopharmaceuticals and may contribute to translational work in people affected by non-Hodgkin lymphoma.


Subject(s)
Lymphoma, Non-Hodgkin/diagnosis , Organotechnetium Compounds/chemistry , Radiopharmaceuticals/chemistry , Animals , Dipeptides/chemistry , Dipeptides/pharmacokinetics , Dipeptides/toxicity , Dogs , Mice , Organotechnetium Compounds/pharmacokinetics , Organotechnetium Compounds/toxicity , Phenylurea Compounds/chemistry , Phenylurea Compounds/pharmacokinetics , Phenylurea Compounds/toxicity , Radiopharmaceuticals/pharmacokinetics , Radiopharmaceuticals/toxicity , Tissue Distribution , Whole Body Imaging
2.
Semin Nucl Med ; 42(3): 147-50, 2012 May.
Article in English | MEDLINE | ID: mdl-22475423

ABSTRACT

Although the term has been coined recently, the concepts underlying theranosis have been applied in patient care for more than one-half century. However, advanced technologies are used now. Theranosis describes processes used to tailor therapy for a patient. It is the use of diagnostic tests to identify those patients better-suited for a drug (or drugs) or to determine how well a drug is working. (131)I-iodide for imaging and for therapy of hyperthyroidism and thyroid cancer is an excellent example of personalized theranosis and has withstood challenge for more than 50 years. Radioimmunotherapy for non-Hodgkin lymphoma is a more recent example of theranosis. Either of 2 anti-CD20 monoclonal antibodies, one labeled with indium for imaging or (90)Y for radiotherapy or a second labeled with (131)I for both imaging and radiotherapy, is used for salvage and first-line therapy of multifocal non-Hodgkin lymphoma. The efficacy of these drugs is greater than that of alternative therapies. To mimic the molecular specificity and cell selectivity of a monoclonal antibody, smaller molecules that also bind to proteins upregulated by malignant cells can be used to transport cytotoxic agents to the malignant cells. Smaller carrier molecules like peptides, aptamers, affibodies, and selective, high-affinity ligands facilitate intensification of therapy because of their size. Personalized genomics, proteomics, and molecular imaging are among technologies currently used for theranosis. Molecular emission tomographic imaging with radiolabeled drugs has been used to examine the pharmacology of anticancer therapies and their effectiveness. Increased glycolysis, a molecular phenotype of many malignancies, can be imaged using (18)F-fluoro-2-deoxyglucose (FDG). Tomographic imaging using FDG allows stratification of patients into those responding and likely to respond to the therapy and those better treated in another manner. Prediction of therapeutic response avoids useless therapy so that FDG imaging is included in official response evaluation criteria. Although a fixed approach to therapy may be more practical, an individualized approach is more likely to ensure that each patient receives an effective drug and drug dose that has acceptable and definable tissue effects. Drugs that work in one individual may be ineffective or cause adverse events in others.


Subject(s)
Lymphoma, Non-Hodgkin/diagnosis , Lymphoma, Non-Hodgkin/therapy , Humans , Iodine Radioisotopes/therapeutic use , Lymphoma, Non-Hodgkin/radiotherapy , Precision Medicine
3.
Org Biomol Chem ; 10(8): 1521-6, 2012 Feb 28.
Article in English | MEDLINE | ID: mdl-22132412

ABSTRACT

Multivalent single chain variable fragments (scFv) show increased affinity to tumor-associated antigens compared to monovalent scFv and intact monoclonal antibodies (mAb). Multivalent constructs can be derived from self-associating or covalent scFv with covalent constructs offering improved in vivo and in vitro stability. Covalent attachment of scFv can be achieved using genetically engineered expression vectors that afford scFv with site specific cysteine functionality. Expression vectors for di-scFv-C wherein the cysteine is located in the center of two scFv have also been developed for attaching chemically reactive linkers. In the example illustrated here, the di-scFv-C is derived from a mAb directed against the MUC1 epitope, which is presented on cancer cells. To achieve multivalency, a chemical crosslinking strategy utilizing various azide and multi-alkyne functionalized polyethylene glycol (PEG) linkers was implemented. Conjugation was achieved by attachment of these linkers to the scFv thiol functionality. Chemoselective ligation was employed to covalently link different protein conjugates via copper(I) catalyzed azide alkyne 1,3-dipolar cycloaddition reaction (CuAAC) chemistry. Ligations were achieved in >70% yield using a specific set of linkers as determined by SDS-PAGE and densitometry. ELISA showed increased tumor binding of a tetravalent scFv providing a versatile chemical crosslinking strategy for construction of multivalent and bi-specific immunoconjugates that retain biological activity and have potential application in pre-targeted radioimmunotherapy and imaging.


Subject(s)
Single-Chain Antibodies/chemistry , Azides/chemistry , Cyclization , Molecular Structure
4.
Cancer Res ; 70(13): 5448-56, 2010 Jul 01.
Article in English | MEDLINE | ID: mdl-20530664

ABSTRACT

Integrin alpha(4)beta(1) is an attractive but poorly understood target for selective diagnosis and treatment of T-cell and B-cell lymphomas. This report focuses on the rapid microwave preparation, structure-activity relationships, and biological evaluation of medicinally pertinent benzimidazole heterocycles as integrin alpha(4)beta(1) antagonists. We documented tumor uptake of derivatives labeled with (125)I in xenograft murine models of B-cell lymphoma. Molecular homology models of integrin alpha(4)beta(1) predicted that docked halobenzimidazole carboxamides have the halogen atom in a suitable orientation for halogen-hydrogen bonding. The high-affinity halogenated ligands identified offer attractive tools for medicinal and biological use, including fluoro and iodo derivatives with potential radiodiagnostic ((18)F) or radiotherapeutic ((131)I) applications, or chloro and bromo analogues that could provide structural insights into integrin-ligand interactions through photoaffinity, cross-linking/mass spectroscopy, and X-ray crystallographic studies.


Subject(s)
Benzimidazoles/pharmacology , Integrin alpha4beta1/antagonists & inhibitors , Lymphoma, B-Cell/drug therapy , Lymphoma, T-Cell/drug therapy , Amino Acid Sequence , Animals , Benzimidazoles/chemistry , Benzimidazoles/pharmacokinetics , Female , Fluorine Radioisotopes/chemistry , Integrin alpha4beta1/metabolism , Iodine Radioisotopes/chemistry , Isotope Labeling , Lymphoma, B-Cell/metabolism , Lymphoma, T-Cell/metabolism , Mice , Mice, Inbred BALB C , Mice, Nude , Models, Molecular , Molecular Sequence Data , Radiopharmaceuticals/chemistry , Radiopharmaceuticals/pharmacokinetics , Radiopharmaceuticals/pharmacology , Structure-Activity Relationship , Xenograft Model Antitumor Assays
5.
Chem Biol Drug Des ; 75(5): 455-60, 2010 May.
Article in English | MEDLINE | ID: mdl-20486931

ABSTRACT

Peptides are labile toward proteolytic enzymes, and structural modifications are often required to prolong their metabolic half-life and increase resistance. One modification is the incorporation of non-alpha-amino acids into the peptide to deter recognition by hydrolytic enzymes. We previously reported the synthesis of chimeric alpha/delta-peptides from glutamic acids (Glu) and the sialic acid derivative Neu2en. Conformational analyses revealed these constructs adopt secondary structures in water and may serve as conformational surrogates of polysialic acid. Polysialic acid is a tumor-associated polysaccharide and is correlated with cancer metastasis. Soluble polysialic acid is rapidly cleared from the blood limiting its potential for vaccine development. One motivation in developing structural surrogates of polysialic acid was to create constructs with increased bioavailability. Here, we report plasma stability profiles of Glu/Neu2en alpha/delta-peptides. DOTA was conjugated at the peptide N-termini by solid phase peptide synthesis, radiolabeled with (111)In, incubated in human blood plasma at 37 degrees C, and their degradation patterns monitored by cellulose acetate electrophoresis and radioactivity counting. Results indicate that these peptides exhibit a long half-life that is two- to three-orders of magnitude higher than natural alpha-peptides. These findings provide a viable platform for the synthesis of plasma stable, sialic acid-derived peptides that may find pharmaceutical application.


Subject(s)
N-Acetylneuraminic Acid/chemistry , Peptides/chemical synthesis , Catalytic Domain , Computer Simulation , Half-Life , Heterocyclic Compounds, 1-Ring/chemistry , Humans , N-Acetylneuraminic Acid/chemical synthesis , Peptides/blood , Peptides/chemistry , Protein Stability , Protein Structure, Secondary , Sialic Acids/chemical synthesis , Sialic Acids/chemistry
6.
Semin Nucl Med ; 40(2): 136-44, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20113681

ABSTRACT

Although most patients with locoregional cancer are cured by surgery, radiotherapy, chemotherapy, and combinations thereof, those with distant metastases are not despite systemic chemotherapy. These patients respond to local radiotherapy but generally need systemic therapy. Non-Hodgkin's lymphoma (NHL) provides a paradigm for the role of molecular targeted radiotherapy (MTRT) because these patients have multifocal disease in most cases. Although patients with NHL achieve remissions after multiple cycles of chemotherapy, less than one half of those with aggressive NHL are cured and almost none of those with low grade NHL. Furthermore, NHL, like other cancers, becomes chemoresistant, yet remains responsive to radiotherapy. MTRT, radiation targeted by molecules, is a good strategy for the treatment of multifocal and radiosensitive cancers. Radioimmunotherapy (RIT) is an MTRT approach using MAbs, or parts thereof, to target the radionuclide that delivers radiation. Two anti-CD20 monoclonal antibodies (MAbs), one labeled with (111)In for imaging or (90)Y for therapy and a second labeled with (131)I for imaging and therapy, have proven effective and safe for MTRT for NHL patients. The importance of the radiation is demonstrated in the data from the randomized pivotal trial of (90)Y-ibritumomab; response rates were distinctly better in the (90)Y-ibritumomab arm than in the rituximab arm. Furthermore, the efficacy of (131)I-tositumomab was greater than that of the same MAb alone in another pivotal trial. Although hematologic toxicity is dose limiting for MTRT, febrile neutropenia is uncommon. MTRT is also not associated with mucositis, hair loss, or persistent nausea or vomiting, unlike chemotherapy. Randomized trials of MTRT in different strategies have not been conducted, but there is evidence of better outcomes, particularly for strategies that provide dose intensification, such as pretargeted MTRT, multiple dosing ("fractionation"), and MTRT with stem cell transplantation (SCT). Pretargeted RIT separates delivery of the targeting molecule from radionuclide delivery, provides dose escalation, and is more effective than direct one-step RIT, although more complicated to implement. Improved drugs and strategies for MTRT have documented potential for better patient outcomes. Smaller radionuclide carriers, such as those used for pretargeted MTRT, should be incorporated into the management of patients with NHL and other cancers soon after the patients have proven incurable. Expected improvements using better drugs, strategies, and combinations with other drugs seem likely to make MTRT integral in the management of many patients with cancer and likely to lead to cures of NHL.


Subject(s)
Lymphoma/radiotherapy , Radiation Dosage , Radiotherapy/methods , Animals , Humans , Immunotherapy , Lymphoma/therapy , Radiometry , Radiotherapy Dosage
8.
Ann N Y Acad Sci ; 1161: 74-82, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19426307

ABSTRACT

Multivalency results from the simultaneous binding of multiple ligands with multiple receptors. Understanding the effect of multivalency on binding kinetics in molecular and cellular systems may aid the development of new types of therapeutics or countermeasures to pathogen infection. Here, we describe a method using single-molecule dynamic force spectroscopy to determine the binding strength of antibody-protein complexes as a function of binding valency in a direct and simple measurement. We used the atomic force microscope to measure the force required to rupture a single complex formed by the MUC1 protein, a cancer indicator, and therapeutic antibodies that target MUC1. We will show that nanomechanical polymer tethers can be used in a new manner to count the number of biological bonds formed. Mechanical work will disrupt these bonds and can be used to quantify the overall kinetics. This ability to form, count, and dissociate biological bonds with nanomechanical forces provides a powerful method to study the physical laws governing the interactions of biological molecules.


Subject(s)
Spectrum Analysis/methods , Elasticity , Kinetics , Mucin-1/analysis , Mucin-1/chemistry
9.
Mol Cancer ; 8: 25, 2009 Apr 22.
Article in English | MEDLINE | ID: mdl-19383174

ABSTRACT

BACKGROUND: A variety of arginine-rich peptide sequences similar to those found in viral proteins have been conjugated to other molecules to facilitate their transport into the cytoplasm and nucleus of targeted cells. The selective high affinity ligand (SHAL) (DvLPBaPPP)2LLDo, which was developed to bind only to cells expressing HLA-DR10, has been conjugated to one of these peptide transduction domains, hexa-arginine, to assess the impact of the peptide on SHAL uptake and internalization by Raji cells, a B-cell lymphoma. RESULTS: An analog of the SHAL (DvLPBaPPP)2LLDo containing a hexa-arginine peptide was created by adding six D-arginine residues sequentially to a lysine inserted in the SHAL's linker. SHAL binding, internalization and residualization by Raji cells expressing HLA-DR10 were examined using whole cell binding assays and confocal microscopy. Raji cells were observed to bind two fold more 111In-labeled hexa-arginine SHAL analog than Raji cells treated with the parent SHAL. Three fold more hexa-arginine SHAL remained associated with the Raji cells after washing, suggesting that the peptide also enhanced residualization of the 111In transported into cells. Confocal microscopy showed both SHALs localized in the cytoplasm of Raji cells, whereas a fraction of the hexa-arginine SHAL localized in the nucleus. CONCLUSION: The incorporation of a hexa-D-arginine peptide into the linker of the SHAL (DvLPBaPPP)2LLDo enhanced both the uptake and residualization of the SHAL analog by Raji cells. In contrast to the abundant cell surface binding observed with Lym-1 antibody, the majority of (DvLPBaPPP)2LArg6AcLLDo and the parent SHAL were internalized. Some of the internalized hexa-arginine SHAL analog was also associated with the nucleus. These results demonstrate that several important SHAL properties, including uptake, internalization, retention and possibly intracellular distribution, can be enhanced or modified by conjugating the SHALs to a short polypeptide.


Subject(s)
Arginine/metabolism , Heterocyclic Compounds, 1-Ring/metabolism , Lymphoma, B-Cell/metabolism , Oligopeptides/metabolism , Peptides/metabolism , Arginine/chemistry , Binding Sites , Cell Line, Tumor , Cell Nucleus/metabolism , HLA-DR Antigens/metabolism , HLA-DR Serological Subtypes , Heterocyclic Compounds, 1-Ring/chemistry , Humans , Ligands , Microscopy, Confocal , Oligopeptides/chemistry , Peptides/chemistry
10.
J Nucl Med ; 50(4): 625-34, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19289419

ABSTRACT

UNLABELLED: N-[[4-[[[(2-ethylphenyl)amino]carbonyl]amino]phenyl]acetyl]-N(epsilon)-6-[(2E)-1-oxo-3-(3-pyridinyl-2-propenyl)]-l-lysyl-l-2-aminohexanedioyl-(1-amino-1-cyclohexane)carboxamide (LLP2A) is a high-affinity, high-specificity peptidomimetic ligand (inhibitory concentration of 50% = 2 pM) that binds the activated alpha4beta1 integrin found on a variety of malignant lymphoid cell lines. To better determine whether this ligand holds promise for imaging and therapy in lymphoid malignancies, 6 LLP2A derivatives, as LLP2A-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (LLP2A-DOTA) and LLP2A-DOTA-polyethylene glycol (LLP2A-DOTA-PEG), were designed, synthesized, and radiolabeled with (111)In. Comparative pharmacokinetic studies in mice with Raji B-cell lymphoma xenografts were then complemented by small-animal PET of the lead molecular LLP2A format using (64)Cu-LLP2A-11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane ((64)Cu-LLP2A-CB-TE2A). METHODS: LLP2A-DOTA and LLP2A-CB-TE2A were prepared using solid-phase synthesis; LLP2A-DOTA-PEG(2,000), LLP2A-DOTA-PEG(5,000), LLP2A-DOTA-PEG(10,000), (LLP2A-DOTA)(2)PEG(10,000), and (LLP2A-DOTA)(4)PEG(10,000) were prepared by PEGylation. (111)In radiolabeling of DOTA and (64)Cu radiolabeling of CB-TE2A conjugates yielded 370-1,850 and 3,700-7,400 kBq/microg (10-50 and 100-200 microCi/microg), respectively. The pharmacokinetics of the six (111)In radioconjugates were studied in vivo using biodistribution data (4 and 24 h) and whole-body autoradiography (24 h) in mice with Raji tumor xenografts. (64)Cu-LLP2A-CB-TE2A was imaged (4 and 24 h) on a small-animal PET scanner in the same mouse model. RESULTS: The highest tumor uptake in pharmacokinetic studies was obtained with LLP2A-DOTA and (LLP2A-DOTA)(4)-PEG(10,000). For (111)In-LLP2A-DOTA (1 nM) at 4 and 24 h after injection, ratios of tumor to blood and tumor to nontumor (normal) organ (T/NT) were 8 to 35:1 for all organs or tissue except the spleen, marrow, and kidney, which were between 2:1 and 1:1. Tetravalent (LLP2A-DOTA)(4)-PEG(10,000) (1.1 nM) had tumor uptake similar to the univalent LLP2A-DOTA but higher liver, marrow, and kidney uptake. The excellent T/NT of LLP2A was also demonstrated by small-animal PET with (64)Cu-LLP2A-CB-TE2A at both 4 and 24 h after injection; obvious spleen targeting was apparent, but little kidney or liver activity was observed. CONCLUSION: Of the conjugates investigated, the univalent, non-PEGylated ligand (111)In-LLP2A-DOTA exhibited the best T/NT ratios and showed the greatest potential for imaging of alpha4beta1 in human lymphoma. Furthermore, this univalent non-PEGylated LLP2A format, as (64)Cu-LLP2A-CB-TE2A, demonstrated excellent tumor targeting by small-animal PET and warrants further investigation as an agent for the study of alpha4beta1 expression in human lymphoid malignancies.


Subject(s)
Burkitt Lymphoma/diagnostic imaging , Burkitt Lymphoma/metabolism , Integrin alpha4beta1/metabolism , Organometallic Compounds/pharmacokinetics , Polyethylene Glycols/pharmacokinetics , Animals , Burkitt Lymphoma/radiotherapy , Cell Line, Tumor , Drug Delivery Systems/methods , Female , Humans , Metabolic Clearance Rate , Mice , Mice, Inbred C57BL , Mice, Nude , Organ Specificity , Organometallic Compounds/therapeutic use , Polyethylene Glycols/therapeutic use , Radionuclide Imaging , Radiopharmaceuticals/chemical synthesis , Radiopharmaceuticals/pharmacokinetics , Tissue Distribution
11.
Cancer Biother Radiopharm ; 23(4): 383-97, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18771343

ABSTRACT

The cytocidal potency of a molecule can be augmented by conjugating a radionuclide for molecular targeted radionuclide therapy (MTRT) for cancer. Radioimmunotherapy (RIT) should be incorporated into the management of patients with B-cell non-Hodgkin's lymphoma (NHL) soon after the patients have proven incurable. Better drugs, strategies, and combinations with other drugs seem certain to make RIT integral to the management of patients with NHL and likely to lead to a cure of the currently incurable NHL. These improved drugs, strategies, and combinations thereof also offer opportunities for RIT to become part of the management of solid malignancies, including epithelial cancers. Smaller radionuclide carriers, such as those used for pretargeted strategies, provide dose intensification. The potential of pretargeted RIT to improve patient outcomes is striking.


Subject(s)
Lymphoma/radiotherapy , Neoplasms/radiotherapy , Radiotherapy/methods , Animals , Clinical Trials as Topic , Combined Modality Therapy/methods , Dose-Response Relationship, Radiation , Hematopoietic Stem Cell Transplantation , Humans , Lymphoma/therapy , Lymphoma, Non-Hodgkin/radiotherapy , Lymphoma, Non-Hodgkin/therapy , Neoplasms/therapy , Radiation-Sensitizing Agents/therapeutic use , Radioimmunotherapy/adverse effects , Radioimmunotherapy/methods , Radiotherapy/adverse effects , Radiotherapy Dosage , Salvage Therapy/methods
12.
Bioorg Med Chem Lett ; 18(17): 4802-5, 2008 Sep 01.
Article in English | MEDLINE | ID: mdl-18701282

ABSTRACT

Radioimmunotherapy (RIT) is a method for selectively delivering radionuclides to cancer cells while reducing the radiation dose to normal tissues. However, because of slow clearance of MAbs, normal tissues also received radiotoxicity. One of the promising strategies is linking on-demand cleavable (ODC) peptides between radiometal chelates and the tumor targeting agents. We have tested this proof-of-concept by using ODC peptides that are designed to be cleaved only by TNKase and are resistant to cleavage by enzymes present in the plasma and the tumor. TNKase-specific peptide linkers using l- and d-amino acids were screened by OBOC combinatorial peptide libraries. One of the best peptides was linked to radiometal chelate and ChL6-MAb to prepare radioimmunoconjugate (RIC). Optimization and characterization of the linker conjugation to MAb show (a) 1-2 peptides linked to each MAb; (b) immunoreactivity >80%; (c) specific activity of the RIC 0.7-1 microCi/microg; (d) RIC stable over 7 days in human plasma; and (e) radiometal-chelated ODC peptide cleaved from the RIC in plasma by TNKase at clinical dose levels of 10 microg/ml. The percent release of radiochelate from RIC was 50% at 24h and 85% over 7 2h in vitro. This novel ODC-linked RIC could be a potential molecule for RIT.


Subject(s)
Immunoconjugates/therapeutic use , Peptides/therapeutic use , Radioimmunotherapy , Tissue Plasminogen Activator/therapeutic use , Animals , Antibodies, Monoclonal/metabolism , Antibodies, Monoclonal/therapeutic use , Humans , Hydrolysis , Mice , Neoplasms/immunology , Neoplasms/metabolism , Neoplasms/radiotherapy , Peptides/chemistry , Peptides/metabolism , Radioimmunotherapy/methods , Tenecteplase , Tissue Plasminogen Activator/metabolism , Tumor Cells, Cultured
13.
Cancer Biother Radiopharm ; 23(2): 265-71, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18454696

ABSTRACT

UNLABELLED: Nanoparticle thermotherapy (NPTT) uses monoclonal antibody-linked iron oxide magnetic nanoparticles (bioprobes) for the tumor-specific thermotherapy of cancer by hysteretic heating of the magnetic component of the probes through an externally applied alternating magnetic field (AMF). The present study investigated the effect of NPTT on a human prostate cancer cell line, DU145. The concept of total heat dose (THD) as a measure for NPTT was validated on a cellular level and THD was correlated to cell death in vitro. The study, furthermore, explored the potential enhancement of the NPTT effect through added external beam radiation therapy (EBRT), because both forms of treatment have a different, and potentially complementary, mechanism of causing cell death. METHODS: Using carbodiimide, (111)In-DOTA-ChL6 was conjugated to dextran iron oxide 20-nm particles with polyethylene glycol COOH groups on the surface and purified as (111)In-bioprobes. NPTT and EBRT were applied alone and combined to cells labeled with the bioprobes. Cell response was monitored by measuring lactate dehydrogenase (LDH), a product of cytolysis, in the medium. This distinct focus on the response to NPTT was possible, since we found in previous studies that the LDH assay was relatively insensitive to the response of cells (without bioprobes) to EBRT in the dose levels given here. RESULTS: NPTT showed a significantly increased cell death at a total calculated heat dose of 14.51 and 29.02 J/g cells (50% and 100% AMF duty, 350 Oe, 136 kHz, 12 cycles, 20 minutes total), compared with AMF exposure in the absence of bioprobes. Adding EBRT to NPTT did not increase cell death, as measured by LDH. However, EBRT given to cells labeled with bioprobes caused significant cell death at radiation doses of 10 Gy and higher. CONCLUSIONS: In human prostate cancer cell cultures, NPTT applied as a single modality caused cell death that correlated with THD estimation; complete cell death occurred at 14.51 J/g cells. Consequently, enhancement of the NPTT effect through the addition of EBRT could not be addressed. Interestingly, EBRT induced cell death on bioprobe-labeled cells at EBRT levels that did not show cell death in the absence of bioprobes; this phenomenon is worth investigating further.


Subject(s)
Nanoparticles , Prostatic Neoplasms/pathology , Cell Line, Tumor , Cell Survival/radiation effects , Humans , Hyperthermia, Induced , Lactate Dehydrogenases/metabolism , Male , Prostatic Neoplasms/enzymology
14.
Cancer Biother Radiopharm ; 23(1): 82-91, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18298332

ABSTRACT

BACKGROUND: Noninvasive, focused hyperthermia can be achieved by using an externally applied alternating magnetic field (AMF) if effective concentrations of nanoparticles can be delivered to the target cancer cells. Targeting agents, for example, monoclonal antibodies or peptides, linked to magnetic iron oxide nanoparticles (NP), represent a promising strategy to target cancer cells and hyperthermia. METHODS: We have developed a new radioconjugate NP ((111)In-DOTA-di-scFv-NP), using recombinantly generated antibody fragments, di-scFv-c, for the imaging and therapy of anti-MUC-1-expressing cancers, because aberrant MUC-1 is abundantly expressed on the majority of human epithelial cancers. Anti-MUC-1 di-scFv-c (50 kDa) were engineered, generated, and selected to link maleimide functionalized nanoparticles (NP-M). DOTA chelate was conjugated with di-scFv-c for radionuclide chelation to trace the radioimmunonanoparticles (RINPs) in vivo. RESULTS: Heat-inducing NP-M were prepared with maleimide density >15 per particle for site-specific thiolation. The specific activity of the RINP was 4-5 microCi (111)In/mg with >10 molecules of di-scFv per NP. We characterized the RINP by polyacrylamide gel electrophoresis, cellulose acetate electrophoresis, size-exclusion chromatography, and tumor-cell binding. RINP had a >90% di-scFv conjugated to NP and an immunoreactivity >80% relative to unmodified di-scFv-c on HBT 3477 and DU145 tumor cells. Pharmacokinetics and whole-body autoradiography studies demonstrated that a 5% injected dose was targeted in tumor after 24 hours. CONCLUSIONS: Further development of this new preparation of RINP may provide uniquely high tumor-targeting NP for AMF-driven tumor hyperthermia with less spleen and kidney accumulation.


Subject(s)
Mucin-1/metabolism , Nanoparticles/therapeutic use , Neoplasms/diagnostic imaging , Neoplasms/radiotherapy , Radioimmunotherapy , Animals , Antibodies, Monoclonal/therapeutic use , Blotting, Western , COS Cells , Chlorocebus aethiops , Female , Ferric Compounds/chemistry , Ferric Compounds/metabolism , Hot Temperature , Humans , Immunoglobulin Variable Region/immunology , Indium Radioisotopes/pharmacokinetics , Indium Radioisotopes/therapeutic use , Magnetics , Maleimides/chemistry , Maleimides/metabolism , Mice , Mice, Inbred BALB C , Mice, Nude , Mucin-1/genetics , Mucin-1/immunology , Neoplasms/immunology , Radiography , Radioimmunodetection , Tissue Distribution , Tumor Cells, Cultured/metabolism
15.
Cancer Biother Radiopharm ; 23(6): 671-80, 2008 Dec.
Article in English | MEDLINE | ID: mdl-20443694

ABSTRACT

The promise of hyperthermia has yet to be realized, but the fundamental idea and the effects of heat on (cancer) cells are well known. Cell death from exposure to heat is a function of both the intensity of the heat and the length of the exposure. Cells die by necrosis and by apoptosis. Sublethal heat doses sensitize cancer cells to radiation and drugs. Because of advances in chemistry and physics, harnessing the power of heat to kill cancer cells seems achievable now! Using novel systems embodied in the combination of molecular-targeted nanoparticles and hysteretic heating of the nanoparticles with "focused" alternating magnetic frequencies (AMFs), heat delivery can be better controlled. Importantly, hyperthermia does not damage, and may actually enhance, the immune system. Trials in patients are needed to settle the clinical role of new thermal treatment.


Subject(s)
Hyperthermia, Induced , Neoplasms/therapy , Animals , Hot Temperature , Humans
16.
Cancer Biother Radiopharm ; 23(6): 783-96, 2008 Dec.
Article in English | MEDLINE | ID: mdl-20443696

ABSTRACT

To mimic the molecular specificity and cell selectivity of monoclonal antibody (mAb) binding while decreasing size, nanomolecules (selective high-affinity ligands; SHALs), based on in silico modeling, have been created to bind to human leukocyte antigen-DR (HLA-DR10), a signaling receptor protein upregulated on the malignant B-lymphocytes of non-Hodgkin's lymphoma and chronic lymphocytic leukemia. SHALs were synthesized with a biotin or DOTA chelate (1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid), using a solid-phase lysine-polyethyleneglycol backbone to link sets of ligands shown previously to bind to HLA-DR10. Using cell-binding and death assays and confocal microscopy, SHAL uptake, residualization, and cytocidal activity were evaluated in HLA-DR10 expressing and nonexpressing live, human lymphoma cell lines. All of the SHALs tested were selective for, and accumulated in, expressing cells. Reflecting binding to HLA-DR10 inside the cells, SHALs having the Ct ligand (3-(2-([3-chloro-5-trifluoromethyl)-2-pyridinyl]oxy)-anilino)-3-oxopropanionic acid) residualized in expressing cells greater than 179 times more than accountable by cell-surface membrane HLA-DR10. Confocal microscopy confirmed the intracellular residualization of these SHALs. Importantly, SHALs with a Ct ligand had direct cytocidal activity, similar in potency to that of Lym-1 mAb and rituximab, selectively for HLA-DR10 expressing lymphoma cells and xenografts. The results show that SHALs containing the Ct ligand residualize intracellularly and have cytocidal effects mediated by HLA-DR10. These SHALs have extraordinary potential as novel molecules for the selective targeting of lymphoma and leukemia for molecular therapy and imaging. Further, these SHALs can be used to transport and residualize cytotoxic agents near critical sites inside these malignant cells.


Subject(s)
Antibodies, Monoclonal/immunology , Biomimetic Materials/pharmacology , HLA-DR Antigens/immunology , Leukemia/therapy , Lymphoma/therapy , Molecular Mimicry/immunology , Antibodies, Monoclonal/therapeutic use , Antibodies, Monoclonal, Murine-Derived , Biomimetic Materials/chemistry , Cell Line, Tumor , Cell Survival/drug effects , HLA-DR Serological Subtypes , Humans , Indium Radioisotopes , Leukemia/immunology , Lymphoma/immunology , Nanoparticles , Peptide Fragments/chemistry , Peptide Fragments/pharmacology , Radiopharmaceuticals , Rituximab , Spectrometry, Mass, Electrospray Ionization , Tissue Distribution
17.
Cancer Biother Radiopharm ; 22(4): 521-30, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17803447

ABSTRACT

Anti-CD20 monoclonal antibodies (mAbs), unlabeled rituximab (Rituxan, Biogen Idec Inc., Cambridge, MA; and Genentech Inc., South San Francisco, CA) or radiolabeled 90Y-ibritumomab (Zevalin, Biogen Idec Inc., Cambridge, MA) and 131I-tositumomab (Bexxar; Glaxo Smith Kline, Research Triangle Park, NC), have proven to be effective therapy for non-Hodgkin's lymphoma (NHL), but also induce immediate and persistent decreases in normal peripheral blood lymphocytes (PBLs). Lym-1, a mAb that selectively targets malignant lymphocytes, also has induced therapeutic responses and prolonged survival in patients with NHL when labeled with iodine-131 (131I). We have retrospectively examined its effect on PBLs in 41 NHL patients that had received 131I-Lym-1 therapy. Absolute lymphocyte counts (ALCs) were evaluated before and after the first and last 131I-Lym-1 infusion. Modest decreases in PBLs were observed in most of the patients. Using strict criteria to define recovery, time to recovery was determined for 19 patients, with the remainder censored because of insufficient follow-up (median follow up for censored patients: 22 days). Using Kaplan-Meier estimates, it would be predicted that 31% of patients would recover by 28 days and that median time to recovery would be 44 days after the last 131I-Lym-1 infusion. No predictors were found for time to recovery, considering such factors as the administered Lym-1 or 131I dose, spleen volume, or radiation doses to the body, marrow, or spleen. The data suggest that the effect of 131I-Lym-1 on ALC is the result of a nonspecific radiation effect, rather than a specific Lym-1 mAb effect. The shorter time required for ALC recovery after 131I-Lym-1 when compared to that reported for anti-CD20 mAbs, whether radiolabeled or otherwise, is probably related to differing mechanisms for lymphocytotoxicity and lesser Lym-1 antigenic density on normal B-lymphocytes.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Antibodies, Neoplasm/therapeutic use , Lymphocytes/immunology , Lymphocytes/pathology , Lymphoma, Non-Hodgkin/pathology , Lymphoma, Non-Hodgkin/radiotherapy , Adult , Aged , Antibodies, Monoclonal/adverse effects , Antibodies, Monoclonal, Murine-Derived , Antibodies, Neoplasm/adverse effects , Antibodies, Neoplasm/immunology , Cell Survival/drug effects , Female , Humans , Kaplan-Meier Estimate , Lymphoma, Non-Hodgkin/immunology , Male , Middle Aged , Radioimmunotherapy
18.
Cancer Biother Radiopharm ; 22(4): 531-42, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17803448

ABSTRACT

Selective high-affinity ligands (SHALs) were selected as substitutes for monoclonal antibodies (mAbs) to deliver radioisotopes to malignant tumors. Because a SHAL (5 KD) is considerably smaller in comparison to an antibody (150 KD), a significant therapeutic index (TI) enhancement for radioimmunotherapy (RIT) is anticipated. The antibody-antigen (Ab-Ag) model system chosen for the development of SHALs consists of Lym-1, a MAb with proven selectivity in non-Hodgkin's lymphoma (NHL) patients and its well-characterized Ag, the beta subunit of HLA DR10. Whereas Lym-1 is readily available, the subunit of HLA-DR10 is not. Native, heterodimeric (alpha and beta subunits) HLA-DR10 can be purified from Raji cells, which are known to overexpress this Ag. Inconsistent homogeneity between preparations of HLA-DR10 solubilized in the presence of detergents prompted us to express a recombinant form of the beta subunit of HLA-DR10 in Escherichia coli. Negligible production yields (

Subject(s)
HLA-DR Antigens/genetics , HLA-DR Antigens/metabolism , Lymphoma/immunology , Lymphoma/metabolism , Radioimmunotherapy/methods , Amino Acid Sequence , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal, Murine-Derived , Cell Line, Tumor , Circular Dichroism , Cloning, Molecular , Epitopes/immunology , Escherichia coli , Gene Expression , HLA-DR Antigens/chemistry , HLA-DR Antigens/immunology , HLA-DR Serological Subtypes , Humans , Ligands , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/immunology , Protein Subunits/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , Sensitivity and Specificity , Structural Homology, Protein , Substrate Specificity , Surface Plasmon Resonance
19.
Cancer Biother Radiopharm ; 22(4): 556-63, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17803451

ABSTRACT

UNLABELLED: Protease degradable linkers have been proposed to improve the therapeutic index (TI) (i.e., tumor to normal tissue) of molecular targeted radioisotope therapy by reducing unbound radiotargeting agent in the blood and other normal tissues. If the radioisotope is detached from the circulating targeting agent once the radioisotope level in the tumors has been maximized, the success of this system depends on the ability to anticipate a preferred intervention time that will lead to significantly improved TIs. This paper presents a method to predict preferred intervention times and TIs by using pharmacokinetic tracer studies carried out without intervention. METHODS: Pharmacokinetic data for the blood and tumors from tracer doses of 111In-labeled chimeric and mouse monoclonal antibodies in patients and in mice were used as surrogates for corresponding 90Y radioimmunoconjugates. Data were fit with simple pharmacokinetic functions. A set of formulas was then developed to estimate the improvement in therapeutic index and the preferred intervention time, using simple modeling assumptions. RESULTS: A modeled introduction of enzymatic cleavable linkers resulted in an increase in the tumor-to-blood TI by a factor of 3.2-1.6 for the systems analyzed. As expected, the preferred intervention times varied depending on the pharmacokinetic data, but could be predicted based on a priori knowledge of the actual or anticipated pharmacokinetics in the absence of intervention. CONCLUSIONS: These results highlight the potential value of cleavable linkers in substantially increasing the TI, and provide an approach for estimating a preferred intervention time, using actual or predicted pharmacokinetic data obtained without intervention.


Subject(s)
Blood Vessels/enzymology , Indium Radioisotopes/metabolism , Indium Radioisotopes/pharmacokinetics , Peptides/metabolism , Yttrium Radioisotopes/metabolism , Yttrium Radioisotopes/pharmacokinetics , Animals , Cell Line, Tumor , Humans , Indium Radioisotopes/chemistry , Indium Radioisotopes/therapeutic use , Mice , Models, Animal , Models, Biological , Retrospective Studies , Time Factors , Yttrium Radioisotopes/chemistry , Yttrium Radioisotopes/therapeutic use
20.
Clin Cancer Res ; 13(18 Pt 2): 5621s-5628s, 2007 Sep 15.
Article in English | MEDLINE | ID: mdl-17875798

ABSTRACT

PURPOSE: More than two decades of research and clinical trials have shown radioimmunotherapy to be a promising approach for treating various forms of cancer. Lym-1 antibody, which binds selectively to HLA-DR10 on malignant B-cell lymphocytes, has proved to be effective in delivering radionuclides to non-Hodgkin's lymphoma and leukemia. Using a new approach to create small synthetic molecules that mimic the targeting properties of the Lym-1 antibody, a prototype, selective high-affinity ligand (SHAL), has been developed to bind to a unique region located within the Lym-1 epitope on HLA-DR10. EXPERIMENTAL DESIGN: Computer docking methods were used to predict two sets of small molecules that bind to neighboring cavities on the beta subunit of HLA-DR10 surrounding a critical amino acid in the epitope, and the ligands were confirmed to bind to the protein by nuclear magnetic resonance spectroscopy. Pairs of these molecules were then chemically linked together to produce a series of bidentate and bisbidentate SHALs. RESULTS: These SHALs bind with nanomolar to picomolar K(d)'s only to cell lines expressing HLA-DR10. Analyses of biopsy sections obtained from patients also confirmed that SHAL bound to both small and large cell non-Hodgkin's lymphomas mimicking the selectivity of Lym-1. CONCLUSIONS: These results show that synthetic molecules less than 1/50th the mass of an antibody can be designed to exhibit strong binding to subtle structural features on cell surface proteins similar to those recognized by antibodies. This approach offers great potential for developing small molecule therapeutics that target other types of cancer and disease.


Subject(s)
Antibodies, Monoclonal/chemistry , Antineoplastic Agents/pharmacology , Biomimetic Materials/pharmacology , HLA-DR Antigens/metabolism , Lymphoma, Non-Hodgkin/metabolism , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal, Murine-Derived , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Binding Sites , Biomimetic Materials/chemical synthesis , Biomimetic Materials/chemistry , Cell Line, Tumor , Cell Survival/drug effects , Enzyme-Linked Immunosorbent Assay , HLA-DR Antigens/chemistry , HLA-DR Antigens/immunology , HLA-DR Serological Subtypes , Humans , Ligands , Lymphoma, Non-Hodgkin/pathology , Lymphoma, Non-Hodgkin/therapy , Models, Molecular , Streptavidin/metabolism
SELECTION OF CITATIONS
SEARCH DETAIL
...