RESUMO
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.
Assuntos
Anticorpos de Cadeia Única/química , Azidas/química , Ciclização , Estrutura MolecularRESUMO
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.
Assuntos
Linfoma de Burkitt/diagnóstico por imagem , Linfoma de Burkitt/metabolismo , Integrina alfa4beta1/metabolismo , Compostos Organometálicos/farmacocinética , Polietilenoglicóis/farmacocinética , Animais , Linfoma de Burkitt/radioterapia , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos/métodos , Feminino , Humanos , Taxa de Depuração Metabólica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Especificidade de Órgãos , Compostos Organometálicos/uso terapêutico , Polietilenoglicóis/uso terapêutico , Cintilografia , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/farmacocinética , Distribuição TecidualRESUMO
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.
Assuntos
Imunoconjugados/uso terapêutico , Peptídeos/uso terapêutico , Radioimunoterapia , Ativador de Plasminogênio Tecidual/uso terapêutico , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/uso terapêutico , Humanos , Hidrólise , Camundongos , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/radioterapia , Peptídeos/química , Peptídeos/metabolismo , Radioimunoterapia/métodos , Tenecteplase , Ativador de Plasminogênio Tecidual/metabolismo , Células Tumorais CultivadasRESUMO
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.
Assuntos
Anticorpos Monoclonais/química , Antineoplásicos/farmacologia , Materiais Biomiméticos/farmacologia , Antígenos HLA-DR/metabolismo , Linfoma não Hodgkin/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Murinos , Antineoplásicos/síntese química , Antineoplásicos/química , Sítios de Ligação , Materiais Biomiméticos/síntese química , Materiais Biomiméticos/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Antígenos HLA-DR/química , Antígenos HLA-DR/imunologia , Subtipos Sorológicos de HLA-DR , Humanos , Ligantes , Linfoma não Hodgkin/patologia , Linfoma não Hodgkin/terapia , Modelos Moleculares , Estreptavidina/metabolismoRESUMO
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.
Assuntos
Mucina-1/metabolismo , Nanopartículas/uso terapêutico , Neoplasias/diagnóstico por imagem , Neoplasias/radioterapia , Radioimunoterapia , Animais , Anticorpos Monoclonais/uso terapêutico , Western Blotting , Células COS , Chlorocebus aethiops , Feminino , Compostos Férricos/química , Compostos Férricos/metabolismo , Temperatura Alta , Humanos , Região Variável de Imunoglobulina/imunologia , Radioisótopos de Índio/farmacocinética , Radioisótopos de Índio/uso terapêutico , Magnetismo , Maleimidas/química , Maleimidas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mucina-1/genética , Mucina-1/imunologia , Neoplasias/imunologia , Radiografia , Radioimunodetecção , Distribuição Tecidual , Células Tumorais Cultivadas/metabolismoRESUMO
UNLABELLED: Antibody (mAb)-linked iron oxide nanoparticles (bioprobes) provide the opportunity to develop tumor specific thermal therapy (Rx) for metastatic cancer when inductively heated by an externally applied alternating magnetic field (AMF). To evaluate the potential of this Rx, in vivo tumor targeting, efficacy, and predictive radionuclide-based heat dosimetry were studied using (111)In-ChL6 bioprobes (ChL6 is chimeric L6) in a human breast cancer xenograft model. METHODS: Using carbodiimide, (111)In-DOTA-ChL6 (DOTA is dodecanetetraacetic acid) was conjugated to polyethylene glycol-iron oxide-impregnated dextran 20-nm particles and purified as (111)In-bioprobes. (111)In doses of 740-1,110 kBq (20-30 muCi) (2.2 mg of bioprobes) were injected intravenously into mice bearing HBT3477 human breast cancer xenografts. Pharmacokinetic (PK) data were obtained at 1, 2, 3, and 5 d. AMF was delivered 72 h after bioprobe injection at amplitudes of 1,410 (113 kA/m), 1,300 (104 kA/m), and 700 (56 kA/m) oersteds (Oe) at 30%, 60%, and 90% "on" time (duty), respectively, and at 1,050 Oe (84 kA/m) at 50% and 70% duty over the 20-min treatment. Treated and control mice were monitored for 90 d. Tumor total heat dose (THD) from activated tumor bioprobes was calculated for each Rx group using (111)In-bioprobe tumor concentration and premeasured particle heat response to AMF amplitudes. Tumor growth delay was analyzed by Wilcoxon rank sum comparison of time to double, triple, and quintuple tumor volume in each group, and all groups were compared with the controls. RESULTS: Mean tumor concentration of (111)In-bioprobes at 48 h was 14 +/- 2 percentage injected dose per gram; this concentration 24 h before AMF treatment was used to calculate THD. No particle-related toxicity was observed. Toxicity was observed at the highest AMF amplitude-duty combination of 1,300 Oe and 60% over 20 min; 6 of 10 mice died acutely. Tumor growth delay occurred in all of the other groups, correlated with heat dose and, except for the lowest heat dose group, was statistically significant when compared with the untreated group. Electron microscopy showed (111)In-bioprobes on tumor cells and cell death by necrosis at 24 and 48 h after AMF. CONCLUSION: mAb-guided bioprobes (iron oxide nanoparticles) effectively targeted human breast cancer xenografts in mice. THD, calculated using empirically observed (111)In-bioprobe tumor concentration and in vitro nanoparticle heat induction by AMF, correlated with tumor growth delay.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Temperatura Alta/uso terapêutico , Radioisótopos de Índio/uso terapêutico , Magnetismo , Neoplasias Mamárias Experimentais/terapia , Nanopartículas , Animais , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Transplante de NeoplasiasRESUMO
UNLABELLED: Despite their large size, antibodies (Abs) are suitable carriers to deliver systemic radiotherapy, often molecular image-based, for lymphoma and leukemia. Lym-1 Ab has proven to be an effective radioisotope carrier, even in small amounts, for targeting human leukocyte antigen DR (HLA-DR), a surface membrane protein overexpressed on B-cell lymphoma. Pairs of molecules (referred to as ligands), shown by computational and experimental methods to bind to each of 2 sites within the Lym-1 epitopic region, have been linked to generate small (<2 kDa) molecules (referred to as selective high-affinity ligands [SHALs]) to mimic the targeting properties of Lym-1 Ab. METHODS: A lysine-polyethylene glycol (PEG) backbone was used to synthetically link 2 of the following ligands: deoxycholate, 5-leuenkephalin, triiodothyronine, thyronine, dabsyl-L-valine, and N-benzoyl-L-arginyl-4-amino-benzoic acid to generate a series of 13 bidentate SHALs with a biotin or 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) chelate attached to the linker. These SHALs have been assessed for their selectivity in binding to HLA-DR10-expressing cells and for their pharmacokinetics and tissue biodistribution in mice. Biotinylated versions of these SHALs discriminated cell lines positive for HLA-DR10 expression with near-nanomolar affinity. The DOTA versions of 4 SHALs were labeled with (111)In for pharmacokinetic studies in mice with HLA-DR10-expressing malignant Raji xenografts. RESULTS: The bidentate, biotinylated, and DOTA-SHALs were synthesized in high-purity, multimilligram amounts. Mean radiochemical and product yields and purities were 90%, 75%, and 90% at mean specific activities of 3.9 MBq/microg (105 microCi/microg) for the (111)In-labeled SHALs. As expected, rapid blood clearance and tumor targeting were observed. The pharmacokinetics of the SHALs was influenced by the component ligands. Biliary clearance, kidney localization, and serum receptor binding contributed to less favorable tumor targeting. CONCLUSION: A series of SHALs was readily synthesized in multimilligram amounts and showed the expected selective binding in vitro. Better selection of the SHAL components should provide second-generation SHALs with improved properties to fulfill the substantial potential of these novel molecular carriers for targeting.
Assuntos
Anticorpos Monoclonais/metabolismo , Antígenos HLA-DR/imunologia , Linfoma não Hodgkin/diagnóstico por imagem , Compostos Radiofarmacêuticos/farmacocinética , Animais , Anticorpos Monoclonais Murinos , Biotinilação , Feminino , Humanos , Ligantes , Linfoma não Hodgkin/radioterapia , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos/síntese química , Transplante HeterólogoRESUMO
Despite their large size, antibodies have proven to be suitable radioisotope carriers to deliver systemic radiotherapy, often molecular image-based, for lymphoma and leukemia. To mimic antibody (Ab) targeting behavior while decreasing size by 50-100x, a combination of computational and experimental methods were used to generate molecules that bind to unique sites within the HLA-DR epitopic region of Lym-1, an Ab shown effective in patients. Lym-1 Ab mimics (synthetic high afinity ligands; SHALs) were generated and studied in vitro, using live cell binding assays, and/or pharmacokinetic studies over 24 h in xenografted mice given 1 or 20 microg SHAL doses i.v. Multimilligram amounts of each of the dimeric (bis) SHALs were synthesized at high purity, and labeled with indium-111 at high specific activity and purity. These SHALs were selective for HLA-DR and HLA-DR expressing malignant cells and had functional affinities that ranged from 10(-9) M (nanomolar) to 10(-10) M. Blood clearances ranged from 3.6 to 9.5 h and body clearances ranged from 15.2 to 43.0 h for the 6 bis DOTA-SHALs studied in a mouse model for non-Hodgkin's lymphoma (NHL). While localization was shown in Raji NHL xenografts, biodistribution was influenced by 'sinks' for individual ligands of the SHALs. Highly pure, dimeric mimics for HLA-DR Ab were synthesized, biotinylated and radiolabeled, and showed selectivity in vitro. Pharmacokinetic behavior in mice was influenced by the ligands and by the linker length of the dimeric SHALs. Nanomolar or better functional affinity was observed when a suitably long linker was used to connect the two bidentate SHALs. The concept and methodology are of interest because applicable for targeting most proteins; the SHAL synthetic platform is highly efficient and adaptive.
Assuntos
Anticorpos Monoclonais/metabolismo , Antígenos HLA-DR/imunologia , Linfoma não Hodgkin/diagnóstico por imagem , Mimetismo Molecular , Fragmentos de Peptídeos/imunologia , Compostos Radiofarmacêuticos/farmacocinética , Animais , Anticorpos Monoclonais Murinos , Ensaio de Imunoadsorção Enzimática , Feminino , Antígenos HLA-DR/metabolismo , Subtipos Sorológicos de HLA-DR , Humanos , Ligantes , Linfoma não Hodgkin/radioterapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Estrutura Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/metabolismo , Radioimunoterapia , Cintilografia , Transplante HeterólogoRESUMO
Heterofunctional azide and alkyne PEG-linkers have been synthesized and site specifically conjugated to scFv via a reactive thiol functionality; two scFv were coupled by copper catalyzed 1,3-dipolar cycloaddition to make divalent scFv (di-scFv) with an inter-scFv distance defined to provide divalent binding; antigen binding was maintained for the di-scFv construct and increased several times compared to that of the parent scFv; the cycloaddition reaction reported herein represents an important ligation strategy to covalently link macromolecular proteins and retain sensitive structural conformations.
Assuntos
Região Variável de Imunoglobulina/química , Alcinos , Reações Antígeno-Anticorpo , Antígenos de Neoplasias/análise , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Azidas , Linhagem Celular Tumoral , Reagentes de Ligações Cruzadas/química , Dimerização , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Leves de Imunoglobulina/química , Região Variável de Imunoglobulina/uso terapêutico , Imuno-Histoquímica , Masculino , Engenharia de ProteínasRESUMO
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.
Assuntos
Vasos Sanguíneos/enzimologia , Radioisótopos de Índio/metabolismo , Radioisótopos de Índio/farmacocinética , Peptídeos/metabolismo , Radioisótopos de Ítrio/metabolismo , Radioisótopos de Ítrio/farmacocinética , Animais , Linhagem Celular Tumoral , Humanos , Radioisótopos de Índio/química , Radioisótopos de Índio/uso terapêutico , Camundongos , Modelos Animais , Modelos Biológicos , Estudos Retrospectivos , Fatores de Tempo , Radioisótopos de Ítrio/química , Radioisótopos de Ítrio/uso terapêuticoRESUMO
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.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Antineoplásicos/uso terapêutico , Linfócitos/imunologia , Linfócitos/patologia , Linfoma não Hodgkin/patologia , Linfoma não Hodgkin/radioterapia , Adulto , Idoso , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais Murinos , Anticorpos Antineoplásicos/efeitos adversos , Anticorpos Antineoplásicos/imunologia , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Estimativa de Kaplan-Meier , Linfoma não Hodgkin/imunologia , Masculino , Pessoa de Meia-Idade , RadioimunoterapiaRESUMO
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 (Assuntos
Antígenos HLA-DR/genética
, Antígenos HLA-DR/metabolismo
, Linfoma/imunologia
, Linfoma/metabolismo
, Radioimunoterapia/métodos
, Sequência de Aminoácidos
, Anticorpos Monoclonais/imunologia
, Anticorpos Monoclonais Murinos
, Linhagem Celular Tumoral
, Dicroísmo Circular
, Clonagem Molecular
, Epitopos/imunologia
, Escherichia coli
, Expressão Gênica
, Antígenos HLA-DR/química
, Antígenos HLA-DR/imunologia
, Subtipos Sorológicos de HLA-DR
, Humanos
, Ligantes
, Modelos Moleculares
, Dados de Sequência Molecular
, Conformação Proteica
, Subunidades Proteicas/química
, Subunidades Proteicas/genética
, Subunidades Proteicas/imunologia
, Subunidades Proteicas/metabolismo
, Proteínas Recombinantes/química
, Proteínas Recombinantes/genética
, Proteínas Recombinantes/imunologia
, Proteínas Recombinantes/metabolismo
, Sensibilidade e Especificidade
, Homologia Estrutural de Proteína
, Especificidade por Substrato
, Ressonância de Plasmônio de Superfície
RESUMO
MUC1 mucin expressed in epithelial cancer, such as prostate and breast, is aberrantly glycosylated providing unique targets for imaging and therapy. In order to create a broadly applicable construct to target these unique epitopes on metastatic cancer, we selected an antibody fragment (scFv) that binds both synthetic MUC1 core peptide and epithelial cancer cell-expressed MUC1, and developed a recombinant bivalent molecule (di-scFv). Genetically engineered modifications of the di-scFv were constructed to create five molecular versions, each having a free cysteine (di-scFv-c) at different locations for site-specific conjugation. The effects of the engineered cysteine in the varied sites were studied relative to tumor binding and polyethylene glycol-maleimide (PEG-Mal) conjugation (PEGylation). Escherichia coli production as well as binding to MUC1 core peptide, human tumor cell lines and human tumor biopsies, were comparable. However, the location of the engineered cysteine in these di-scFv-c did influence PEGylation efficiency of this free thiol; higher PEGylation efficiency occurred with this cysteine in the inter-scFv linkage. Di-scFv-c PEG, with the cysteine engineered after the fifth amino acid in the linker, was used as an example to demonstrate comparable antigen-binding to non-PEGylated di-scFv-c. In summary, novel anti-MUC1 di-scFv-c molecules can be efficiently produced, purified and conjugated by site-specific PEGylation without loss of immunoreactivity, thus providing flexible multidentate constructs for cancer-targeted imaging and therapy.
Assuntos
Cisteína/metabolismo , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/imunologia , Mucina-1/imunologia , Engenharia de Proteínas , Linhagem Celular Tumoral , Cromatografia de Afinidade , Células Epiteliais/imunologia , Glicosilação , Humanos , Região Variável de Imunoglobulina/isolamento & purificação , Região Variável de Imunoglobulina/metabolismo , Maleimidas/metabolismo , Mucina-1/metabolismo , Polietilenoglicóis/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
Development of tumor targeting pharmaceuticals on a modular platform is an attractive paradigm. Design choices for bispecific (anti-tumor and anti-chelate) pretargeting molecules are increased by the use of scFvs. Because a scFv is monovalent and small in size, its functional affinity and in vivo residence time can be improved through multimerization. ScFv multimers can be covalent or non-covalent. In vivo studies indicate that covalent scFv multimers are preferable. Attachment of scFv modules to scaffolds offers a wide range of possibilities for size and valency. A free thiol introduced at the C terminal end of a scFv (scFv-SH) allows for site-specific covalent attachment to a PEG scaffold without interfering with its antigen (Ag) binding. Although in theory, multimerization of 3 or 4 scFvs can be achieved by direct conjugation, as scFv-SH, to a tri or tetrafunctionalized PEG, it is not a practical option since homogeneous tri and tetrafunctionalized PEG are not readily available. However, the generation of (scFv)(3-4)-PEG molecules through attachment of combinations of di-scFv-SH (tandemly expressed scFvs) and scFv-SH or 2 di-scFv-SH to a bifunctional PEG is a sound approach that also allows for better control of the scFv-PEG conjugate molecular composition. Optimization of the molecular format of the di-scFv-SH module for production as soluble proteins in E. coli, Ag binding and conjugation is reported in this study. ScFvs in the VH-VL format were used for the di-scFv constructs since Fv domain inversion to VL-VH, while not yielding more protein, also abolished Ag binding. The effects on production yield, Ag binding and conjugation potential of the scFv joining linker length and the presence and location of an engineered cysteine were assessed in vitro. Our data indicate that for di-scFv-SH, an increase of the scFv joining linker length results in higher production and better Ag binding; a 20 aa long linker (G(4)S)(4) was the longest linker tested. For the engineered cysteine, three locations were tested; within the scFv joining linker, at the C terminus upstream of the E Tag and as the carboxy terminal aa. The accessibility of the free SH assessed by conjugation of di-scFv-SH to HRP-Mal demonstrated that di-scFv-HRP conjugates are formed with comparable efficiencies when the cysteine is located at the scFv carboxy end. This empirical work provides a framework for the development of bispecific scFv multimers via site-specific attachment of scFv-SH and di-scFv-SH modules to a scaffold.
Assuntos
Cisteína/imunologia , Fragmentos de Imunoglobulinas/imunologia , Engenharia de Proteínas/métodos , Compostos de Sulfidrila/imunologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação de Anticorpos , Western Blotting , Clonagem Molecular , Cisteína/química , Cisteína/genética , Escherichia coli/genética , Fragmentos de Imunoglobulinas/química , Fragmentos de Imunoglobulinas/genética , Camundongos , Dados de Sequência Molecular , Mutagênese Insercional , Polietilenoglicóis/química , Reação em Cadeia da Polimerase , Compostos de Sulfidrila/químicaRESUMO
Although radioimmunotherapy (RIT) has been effective in non-Hodgkin's lymphoma (NHL) as a single agent, solid tumors have shown less clinically significant therapeutic response to RIT alone. The clinical impact of RIT or other forms of targeted radionuclide therapy for solid tumors depends on the development of a high therapeutic index (TI) for the tumor vs. normal tissue effect, and the implementation of RIT as part of synergistic combined modality therapy (CMRIT). Preclinical and clinical studies have provided a wealth of information, and new prototypes or paradigms have shed light on future possibilities in many instances. Evidence suggests that combination and sequencing of RIT in CMRIT appropriately can provide effective treatment for many solid tumors. Vascular targets provide RIT enhancement opportunities and nanoparticles may prove to be effective carriers for RIT combined with intracellular drug delivery or alternating magnetic frequency (AMF) induced thermal tumor necrosis. The sequence and timing of combined modality treatments will be of critical importance to achieve synergy for therapy while minimizing toxicity. Fortunately, the radionuclide used for RIT also provides a signal useful for nondestructive quantitation of the influence of sequence and timing of CMRIT on events in animals and patients. This can be readily accomplished clinically using quantitative high-resolution imaging (e.g., positron emission tomography [PET]).
Assuntos
Neoplasias/radioterapia , Radioimunoterapia/métodos , Neoplasias da Mama/radioterapia , Neoplasias do Colo/radioterapia , Feminino , Humanos , Masculino , Neoplasias/irrigação sanguínea , Neoplasias da Próstata/radioterapia , Dosagem RadioterapêuticaRESUMO
The most potent method for augmenting the cytocidal power of monoclonal antibody (MAb) treatment is to conjugate radionuclides to the MAb to deliver systemic radiotherapy (radioimmunotherapy; RIT). The antigen, MAb, and its epitope can make a difference in the performance of the drug. Additionally, the radionuclide, radiochemistry, chelator for radiometals and the linker between the MAb and chelator can have a major influence on the performance of drugs (radiopharmaceuticals) for RIT. Smaller radionuclide carriers, such as antibody fragments and mimics, and those used for pretargeting strategies, have been described and evaluated. All of these changes in the drugs and strategies for RIT have documented potential for improved performance and patient outcomes. RIT is a promising new therapy that should be incorporated into the management of patients with B-cell non-Hodgkin's lymphoma (NHL) soon after these patients have proven incurable. Predictable improvements using better drugs, strategies, and combinations with other drugs seem certain to make RIT integral to the management of patients with NHL, and likely lead to cure of currently incurable NHL.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Linfoma não Hodgkin/radioterapia , Radioimunoterapia/métodos , Anticorpos Monoclonais/efeitos adversos , Ensaios Clínicos Fase III como Assunto , Humanos , Linfoma de Células B/radioterapia , Compostos Radiofarmacêuticos/uso terapêutico , Dosagem Radioterapêutica , Radioisótopos de Ítrio/uso terapêuticoRESUMO
PURPOSE: Monoclonal antibodies (mAb) in combination and mAbs combined with a radionuclide (radioimmunotherapy) have both been more effective in patients than mAb monotherapy. EXPERIMENTAL DESIGN: Using assays of cell growth and viability, the dose response and temporal characteristics of CD20 (rituximab) and HLA-DR (Lym-1) mAbs, singly and in combination, and of 90Y-conjugated Lym-1 mAb have been characterized in five human lymphoma cell lines (B35M, Raji, SU-DHL-4, SU-DHL-6, and Ramos) spanning Burkitt's to diffuse large cell lymphoma. Although Ramos had a lower HLA-DR density, these cell lines were otherwise selected because of high cell surface CD20 and HLA-DR abundance. Assays of cell growth and death were done using microscopy and trypan blue dye. RESULTS: Lym-1 and rituximab, used singly, showed direct antilymphoma effects; those of Lym-1 were often more potent than those of rituximab. Combinations of these mAbs were more effective, sometimes synergistic, than either mAb singly, even in more resistant SU-DHL-4 cells. Conjugation of 90Y to Lym-1 also augmented potency in all cell lines and overcame resistance to both Lym-1 and rituximab in Ramos cells. CONCLUSIONS: Lym-1 exhibited substantially greater direct antilymphoma effects than rituximab in lymphoma cells in culture. Combination of Lym-1 with rituximab or 90Y increased potency and overcame treatment resistance in lymphoma cells. Greater use of combination therapies of this type to increase potency and range of effectiveness seems likely to improve patient outcome.
Assuntos
Antígenos CD20/biossíntese , Antígenos HLA-DR/química , Antígenos HLA-DR/imunologia , Linfoma/terapia , Radioimunoterapia/métodos , Animais , Anticorpos Monoclonais/química , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Corantes/farmacologia , Terapia Combinada/métodos , Relação Dose-Resposta Imunológica , Humanos , Linfoma Difuso de Grandes Células B/terapia , Camundongos , Microscopia de Fluorescência , Compostos Radiofarmacêuticos/farmacologia , Fatores de Tempo , Resultado do Tratamento , Azul Tripano/farmacologia , Radioisótopos de Ítrio/farmacologiaRESUMO
OBJECTIVE: Magnetic nanoparticles conjugated to a monoclonal antibody can be i.v. injected to target cancer tissue and will rapidly heat when activated by an external alternating magnetic field (AMF). The result is necrosis of the microenvironment provided the concentration of particles and AMF amplitude are sufficient. High-amplitude AMF causes nonspecific heating in tissues through induced eddy currents, which must be minimized. In this study, application of high-amplitude, confined, pulsed AMF to a mouse model is explored with the goal to provide data for a concomitant efficacy study of heating i.v. injected magnetic nanoparticles. METHODS: Thirty-seven female BALB/c athymic nude mice (5-8 weeks) were exposed to an AMF with frequency of 153 kHz, and amplitude (400-1,300 Oe), duration (1-20 minutes), duty (15-100%), and pulse ON time (2-1,200 seconds). Mice were placed in a water-cooled four-turn helical induction coil. Two additional mice, used as controls, were placed in the coil but received no AMF exposure. Tissue and core temperatures as the response were measured in situ and recorded at 1-second intervals. RESULTS: No adverse effects were observed for AMF amplitudes of < or = 700 Oe, even at continuous power application (100% duty) for up to 20 minutes. Mice exposed to AMF amplitudes in excess of 950 Oe experienced morbidity and injury when the duty exceeded 50%. CONCLUSION: High-amplitude AMF (up to 1,300 Oe) was well tolerated provided the duty was adjusted to dissipate heat. Results presented suggest that further tissue temperature regulation can be achieved with suitable variations of pulse width for a given amplitude and duty combination. These results suggest that it is possible to apply high-amplitude AMF (> 500 Oe) with pulsing for a time sufficient to treat cancer tissue in which magnetic nanoparticles have been embedded.
Assuntos
Campos Eletromagnéticos , Temperatura Alta , Nanoestruturas/química , Neoplasias/terapia , Animais , Temperatura Corporal , Feminino , Calefação , Hipertermia Induzida , Magnetismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Temperatura , Fatores de TempoRESUMO
OBJECTIVES: (111)In-chimeric L6 (ChL6) monoclonal antibody (mAb)-linked iron oxide nanoparticle (bioprobes) pharmacokinetics, tumor uptake, and the therapeutic effect of inductively heating these bioprobes by externally applied alternating magnetic field (AMF) were studied in athymic mice bearing human breast cancer HBT 3477 xenografts. Tumor cell radioimmunotargeting of the bioprobes and therapeutic and toxic responses were determined. METHODS: Using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide HCl, (111)In-7,10-tetra-azacyclododecane-N, N',N'',N'''-tetraacetic acid-ChL6 was conjugated to the carboxylated polyethylene glycol on dextran-coated iron oxide 20 nm particles, one to two mAbs per nanoparticle. After magnetic purification and sterile filtration, pharmacokinetics, histopathology, and AMF/bioprobe therapy were done using (111)In-ChL6 bioprobe doses (20 ng/2.2 mg ChL6/ bioprobe), i.v. with 50 microg ChL6 in athymic mice bearing HBT 3477; a 153 kHz AMF was given 72 hours postinjection for therapy with amplitudes of 1,300, 1,000, or 700 Oe. Weights, blood counts, and tumor size were monitored and compared with control mice receiving nothing, or AMF or bioprobes alone. RESULTS: (111)In-ChL6 bioprobe binding in vitro to HBT 3477 cells was 50% to 70% of that of (111)In-ChL6. At 48 hours, tumor, lung, kidney, and marrow uptakes of the (111)In-ChL6 bioprobes were not different from that observed in prior studies of (111)In-ChL6. Significant therapeutic responses from AMF/bioprobe therapy were shown with up to eight times longer mean time to quintuple tumor volume with therapy compared with no treatment (P = 0.0013). Toxicity was only seen in the 1,300 Oe AMF cohort, with 4 of 12 immediate deaths and skin erythema. Electron micrographs showed bioprobes on the surfaces of the HBT 3477 cells of excised tumors and tumor necrosis 24 hours after AMF/bioprobe therapy. CONCLUSION: This study shows that mAb-conjugated nanoparticles (bioprobes), when given i.v., escape into the extravascular space and bind to cancer cell membrane antigen, so that bioprobes can be used in concert with externally applied AMF to deliver thermoablative cancer therapy.
Assuntos
Anticorpos Monoclonais/química , Técnicas Biossensoriais , Radioisótopos de Índio/uso terapêutico , Neoplasias/metabolismo , Neoplasias/terapia , Radioimunoterapia/métodos , Animais , Quelantes/farmacologia , Estudos de Coortes , Campos Eletromagnéticos , Compostos Heterocíclicos com 1 Anel/química , Imunoconjugados/metabolismo , Camundongos , Camundongos Nus , Microscopia Eletrônica , Transplante de Neoplasias , Neoplasias/patologia , Polietilenoglicóis/química , Fatores de Tempo , Distribuição TecidualRESUMO
PURPOSE: New strategies that target selected molecular characteristics and result in an effective therapeutic index are needed for metastatic, hormone-refractory prostate cancer. EXPERIMENTAL DESIGN: A series of preclinical and clinical studies were designed to increase the therapeutic index of targeted radiation therapy for prostate cancer. (111)In/90Y-monoclonal antibody (mAb), m170, which targets aberrant sugars on abnormal MUC1, was evaluated in androgen-independent prostate cancer patients to determine the maximum tolerated dose and efficacy of nonmyeloablative radioimmunotherapy and myeloablative combined modality radioimmunotherapy with paclitaxel. To enhance the tumor to liver therapeutic index, a cathepsin degradable mAb linkage ((111)In/90Y-peptide-m170) was used in the myeloablative combined modality radioimmunotherapy protocol. For tumor to marrow therapeutic index improvement in future studies, anti-MUC1 scFvs modules were developed for pretargeted radioimmunotherapy. Anti-MUC1 and anti-DOTA scFvs were conjugated to polyethylene glycol scaffolds tested on DU145 prostate cancer cells and prostate tissue arrays, along with mAbs against MUC1 epitopes. RESULTS: The nonmyeloablative maximum tolerated dose of 90Y-m170 was 0.74 GBq/m2 for patients with not more than 10% axial skeleton involvement. Metastatic prostate cancer was targeted in all 17 patients; mean radiation dose was 10.5 Gy/GBq and pain response occurred in 7 of 13 patients reporting pain. Myeloablative combined modality radioimmunotherapy with 0.4 GBq/m2 of 90Y-peptide-m170 and paclitaxel showed therapeutic effects in 4 of 6 patients and 30% less radiation to the liver per unit of activity. Neutropenia was dose limiting without marrow support and patient eligibility was a major limitation to dose escalation. Hypoglycosylated MUC1 epitopes were shown to be abundant in prostate cancer and to increase with disease grade. Anti-MUC1 scFvs binding to prostate cancer tissue and live cells were developed into di-scFv binding modules. CONCLUSIONS: The therapeutic index enhancement for prostate radioimmunotherapy was achieved in clinical studies by the addition of cathepsin cleavable linkers to 90Y-conjugated mAbs and the use of paclitaxel. However, the need for marrow support in myeloablative combined modality radioimmunotherapy restricted eligible patients. Therefore, modular pretargeted radioimmunotherapy, aiming at improving the tumor to marrow therapeutic index, is being developed.