RESUMEN
A great deal of interest has surrounded the activities of monoclonal antibodies (mAbs), and mAb-drug, toxin and radionuclide conjugates for the treatment of human cancers. In the last few years, a number of new mAb-based reagents have been clinically approved (Rituxan, Herceptin, and Panorex), and several others are now in advanced clinical trials. Successful therapeutic treatment of solid tumors with drug conjugates of such macromolecules must overcome the barriers to penetration within tumor masses, antigen heterogeneity, conjugated drug potency, and efficient drug release from the mAbs inside tumor cells. An alternative strategy for drug delivery involves a two-step approach to cancer therapy in which mAbs are used to localize enzymes to tumor cell surface antigens. Once the conjugate binds to the cancer cells and clears from the systemic circulation, antitumor prodrugs are administered that are catalytically converted to active drugs by the targeted enzyme. The drugs thus released are capable of penetrating within the tumor mass and eliminating both cells that have and have not bound the mAb-enzyme conjugate. Significant therapeutic effects have been obtained using a broad range of enzymes along with prodrugs that are derived from both approved anticancer drugs and highly potent experimental agents. This review focuses on the activities of several mAb-enzyme/prodrug combinations, with an emphasis on those that have provided mechanistic insight, clinical activity, novel protein constructs, and the potential for reduced immunogenicity.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Enzimas/farmacología , Profármacos/farmacología , Animales , Anticuerpos Monoclonales/química , Antineoplásicos/metabolismo , Biotransformación , Enzimas/química , Humanos , Profármacos/metabolismoRESUMEN
CPT-11 is a clinically approved anticancer drug used for the treatment of advanced colorectal cancer. Upon administration, the carbamate side chain of the drug is hydrolyzed, resulting in the release of SN-38, an agent that has approximately 1000-fold increased cytotoxic activity. Since only a very small percentage of the injected dose of CPT-11 is converted to SN-38, there is a significant opportunity to improve its therapeutic efficacy and to diminish its systemic toxicity by selectively activating the drug within tumor sites. We envisioned that a mAb-human enzyme conjugate for CPT-11 activation would be of interest, particularly since the conjugate would likely be minimally immunogenic, and the prodrug is clinically approved. Toward this end, it was necessary to identify the most active human enzyme that could convert CPT-11 to SN-38. We isolated enzymes from human liver microsomes based on their abilities to effect the conversion and identified human carboxylesterase 2 (hCE-2) as having the greatest specific activity. hCE-2 was 26-fold more active than human carboxylesterase 1 and was 65% as active as rabbit liver carboxylesterase, the most active CPT-11 hydrolyzing enzyme known. The anti-p97 mAb 96.5 was linked to hCE-2, forming a conjugate that could bind to antigen-positive cancer cells and convert CPT-11 to SN-38. Cytotoxicity assays established that the conjugate led to the generation of active drug, but the kinetics of prodrug activation (48 pmol x min(-1) x mg(-1) was insufficient for immunologically specific prodrug activation. These results confirm the importance of hCE-2 for CPT-11 activation and underscore the importance of enzyme kinetics for selective prodrug activation.
Asunto(s)
Antineoplásicos Fitogénicos/metabolismo , Camptotecina/análogos & derivados , Camptotecina/metabolismo , Hidrolasas de Éster Carboxílico/aislamiento & purificación , Sistemas de Liberación de Medicamentos , Profármacos/metabolismo , Secuencia de Aminoácidos , Anticuerpos Antineoplásicos/química , Secuencia de Bases , Unión Competitiva , Hidrolasas de Éster Carboxílico/química , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Inmunoconjugados/metabolismo , Irinotecán , Hígado/enzimología , Datos de Secuencia Molecular , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
To investigate the feasibility of repeated gene transfection in suicide gene therapy against human solid tumors by a combination of 5- fluorocytosine (5-FC) and its converting enzyme, cytosine deaminase (CD), we repeatedly transfected the yeast CD gene into the human pancreatic cancer cell line BXPC3 using the hemagglutinating virus of Japan-liposome in a new gene transfer method. The in vivo growth of the s.c. transplanted BXPC3 tumor in nude mice given CD-gene transfection was significantly suppressed by i.p. injection of 5-FC when compared with tumors treated with the control vector. Furthermore, the tumor transfected with the CD gene during a 7-day interval was suppressed much more than that of a single transfection. These results suggest that repeated transfection of the suicide gene together with the combination of 5-FC and the yeast CD gene using the hemagglutinating virus of Japan-liposome gene transfer method may be useful for the treatment of human solid tumors, including pancreatic cancer.
Asunto(s)
Terapia Genética/métodos , Nucleósido Desaminasas/genética , Neoplasias Pancreáticas/terapia , Respirovirus/genética , Animales , Antimetabolitos Antineoplásicos/farmacocinética , Antimetabolitos Antineoplásicos/farmacología , División Celular/efectos de los fármacos , Citosina Desaminasa , Estudios de Factibilidad , Femenino , Flucitosina/farmacocinética , Flucitosina/farmacología , Fluorouracilo/farmacocinética , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Humanos , Inyecciones Intralesiones , Liposomas , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Nucleósido Desaminasas/biosíntesis , Nucleósido Desaminasas/metabolismo , Neoplasias Pancreáticas/patología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoAsunto(s)
Antibióticos Antineoplásicos/farmacología , Anticuerpos Monoclonales/farmacología , Antígenos de Neoplasias/inmunología , Carcinoma/inmunología , Doxorrubicina/farmacología , Inmunotoxinas/farmacología , Antígenos del Grupo Sanguíneo de Lewis/inmunología , Animales , Antibióticos Antineoplásicos/administración & dosificación , Antibióticos Antineoplásicos/efectos adversos , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/uso terapéutico , Anticuerpos Monoclonales/efectos adversos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Carcinoma/tratamiento farmacológico , Ensayos Clínicos Fase I como Asunto , Neoplasias del Colon/tratamiento farmacológico , Daunorrubicina/química , Daunorrubicina/farmacología , Sistema Digestivo/inmunología , Doxorrubicina/efectos adversos , Doxorrubicina/química , Doxorrubicina/inmunología , Doxorrubicina/uso terapéutico , Diseño de Fármacos , Enfermedades Gastrointestinales/inducido químicamente , Humanos , Inmunotoxinas/efectos adversos , Inmunotoxinas/química , Inmunotoxinas/uso terapéutico , Ratones , Ratones Desnudos , Neoplasias/tratamiento farmacológico , Neoplasias Experimentales/tratamiento farmacológico , Especificidad de Órganos , Ratas , Ratas Endogámicas BN , Ratas Desnudas , Insuficiencia del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Conjugates of the L49 monoclonal antibody (binds to the p97 antigen on melanomas and carcinomas) were formed by attaching Enterobacter cloacae beta-lactamase (bL) to the L49-Fab' fragment using a heterobifunctional cross-linking reagent or by linking the enzyme to L49-sFv using DNA recombinant technology. The conjugates thus formed, L49-Fab'-bL and L49-sFv-bL, were designed to activate cephalosporin containing anticancer prodrugs at the surfaces of antigen positive tumor cells. Results from in vitro experiments using two lung carcinoma cell lines demonstrated that the conjugates were equally active in effecting the release of phenylenediamine mustard from the cephalosporin nitrogen mustard prodrug CCM. While treatment with either of the conjugates combined with the maximum tolerated doses of CCM led to cures of established SN12P renal cell carcinoma tumors in nude mice, only the L49-sFv-bL conjugate maintained its ability to do so at 1/4 the maximum tolerated dose of CCM. L49-sFv-bL was also superior to L49-Fab'-bL in the 1934J renal cell carcinoma tumor model and was shown to be quite active in two in vivo models of human lung carcinoma. These results demonstrate that the recombinant fusion protein leads to more pronounced therapeutic windows than the chemical conjugate and is active in an array of human tumor models.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Antineoplásicos/metabolismo , Profármacos/metabolismo , beta-Lactamasas/química , beta-Lactamasas/genética , Animales , Anticuerpos Monoclonales/metabolismo , Antígenos de Neoplasias/análisis , Antígenos de Neoplasias/metabolismo , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/inmunología , Cefalosporinas/metabolismo , Cefalosporinas/uso terapéutico , Humanos , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/inmunología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/inmunología , Melanoma/inmunología , Antígenos Específicos del Melanoma , Ratones , Ratones Desnudos , Proteínas de Neoplasias/análisis , Proteínas de Neoplasias/metabolismo , Trasplante de Neoplasias , Compuestos de Mostaza Nitrogenada/metabolismo , Compuestos de Mostaza Nitrogenada/uso terapéutico , Proteínas Recombinantes , Células Tumorales CultivadasRESUMEN
Considerable research has been aimed at improving the efficacy of chemotherapeutic agents for cancer therapy. A promising two-step approach that is designed to minimize systemic drug toxicity while maximizing activity in tumours employs monoclonal antibody (mAb)-enzyme conjugates for the activation of anticancer prodrugs. We present, analyse and numerically simulate a mathematical model based on the biology of the system to study the biodistribution, pharmacokinetics and localization properties of mAb-enzyme conjugates in tumour tissue. The model predictions were compared with experimental observations and an excellent correlation was found to exist. In addition, the critical parameters affecting conjugate half-life were determined to be the inter-capillary half-distance and the antibody-antigen binding affinity. An approximation is presented relating the per cent injected dose per gram to inter-capillary half-distance and time. Finally, the model was used to examine various dosing strategies in an attempt to determine which regimen would provide the best biodistribution results. We compared the results of administering a uniform dose of fusion protein via bolus injection, multiple injections and continuous infusion. The model predicts that dosing strategy has little effect on the amount of conjugate that localizes in the tumour.
Asunto(s)
Inmunoglobulina G/metabolismo , Inmunoglobulina G/uso terapéutico , Melanoma/tratamiento farmacológico , Modelos Biológicos , Neoplasias/tratamiento farmacológico , beta-Lactamasas/metabolismo , beta-Lactamasas/uso terapéutico , Animales , Anticuerpos Monoclonales/farmacocinética , Femenino , Humanos , Cinética , Melanoma/inmunología , Melanoma/metabolismo , Ratones , Ratones Desnudos , Modelos Estadísticos , Neoplasias/inmunología , Neoplasias/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacocinética , Proteínas Recombinantes de Fusión/uso terapéutico , Distribución Tisular , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
The monitoring of antibody-directed enzyme-prodrug therapies requires evaluation of drug activation within the tissues of interest. We have demonstrated the feasibility of noninvasive magnetic resonance spectroscopy and spectroscopic imaging (chemical shift imaging) to detect activation of the prodrug 5-fluorocytosine (5-FCyt) to the cytotoxic species 5-fluorouracil (5-FU) by monoclonal antibody-cytosine deaminase (CD) conjugates. In vitro, L6-CD but not 1F5-CD selectively metabolized 5-FCyt to 5-FU on H2981 human lung adenocarcinoma cells because of the presence and absence of cell surface L6 and CD20 antigens, respectively. After pretreatment of H2981 tumor-bearing mice with L6-CD, in vivo metabolism of 5-FCyt to 5-FU within the tumors was detected by 19F magnetic resonance spectroscopy; the chemical shift separation between 5-FCyt and 5-FU resonances was approximately 1.2 ppm. 5-FU levels were 50-100% of 5-FCyt levels in tumors 10-60 min after 5-FCyt administration. Whole body 19F chemical shift imaging (6 x 6 mm in-plane resolution) of tumor-bearing mice demonstrated the highest signal intensity of 5-FU within the tumor region. This study supports further development of noninvasive magnetic resonance methods for preclinical and clinical monitoring of CD enzyme-prodrug therapies.
Asunto(s)
Flucitosina/metabolismo , Fluorouracilo/metabolismo , Profármacos/metabolismo , Animales , Femenino , Flúor , Humanos , Espectroscopía de Resonancia Magnética , RatonesRESUMEN
A panel of 13 renal cell carcinoma cell lines was evaluated for the expression of antigens recognized by the L6 and L49 monoclonal antibodies. All of the cell lines were strongly positive for the L6 antigen, and 9/13 bound 96.5, which, like the L49 monoclonal antibody, recognizes the p97 melanotransferrin antigen. The L6 and L49 antibodies were chemically conjugated to Enterobacter cloacae beta-lactamase (bL), and their abilities to effect site-selective anticancer prodrug activation on two of the renal cell carcinoma cell lines (SN12P and 1934J) were evaluated in vitro and in vivo. L49-bL was 10-90-fold more potent in vitro than L6-bL for the activation of 7-(4-carboxybutanamido)cephalosporin mustard (CCM), a cephalosporin prodrug of phenylenediamine mustard (PDM). In addition, L49-bL showed higher degrees of specific SN12P and 1934J intratumoral uptake than L6-bL, even though the expression of L6 antigen was 2-fold higher than that of p97. These differences might be due to the high-affinity antigen binding of L49-bL relative to L6-bL. In vivo studies utilizing nude mice with established subcutaneous SN12P and 1934J tumor xenografts demonstrated that L49-bL/CCM combinations led to regressions and cures at well-tolerated doses, while L6-bL/CCM and the nonbinding control conjugate P1.17-bL in combination with CCM were ineffective. Conjugate localization in 1934J tumors was much lower than that observed in SN12P tumors, a finding that might acount for the higher activities of L49-bL/CCM in the latter model. These data show that the p97 antigen on renal cell carcinomas can be exploited for selective prodrug activation, even on tumors that localize very small amounts of the L49-bL conjugate.
Asunto(s)
Antineoplásicos/administración & dosificación , Carcinoma de Células Renales/tratamiento farmacológico , Cefalosporinas/administración & dosificación , Inmunoconjugados/uso terapéutico , Neoplasias Renales/tratamiento farmacológico , Compuestos de Mostaza Nitrogenada/administración & dosificación , Profármacos/administración & dosificación , beta-Lactamasas/administración & dosificación , Animales , Anticuerpos Monoclonales , Antígenos de Neoplasias/inmunología , Antígenos de Superficie/inmunología , Antineoplásicos/farmacocinética , Carcinoma de Células Renales/inmunología , Cefalosporinas/farmacocinética , Ensayos de Selección de Medicamentos Antitumorales , Enterobacter cloacae/enzimología , Humanos , Neoplasias Renales/inmunología , Antígenos Específicos del Melanoma , Ratones , Ratones Desnudos , Proteínas de Neoplasias/inmunología , Trasplante de Neoplasias , Compuestos de Mostaza Nitrogenada/farmacocinética , Profármacos/farmacocinética , Trasplante Heterólogo , Células Tumorales Cultivadas , beta-Lactamasas/metabolismoRESUMEN
The synthesis of C-Mel, a cephalosporin carbamate derivative of the clinically used alkylating agent melphalan, is described. C-Mel was designed as an anticancer nitrogen mustard prodrug that releases melphalan upon tumor-specific activation by targeted beta-lactamase (bL). The Km and kcat values for bL hydrolysis of C-Mel were 218 microM and 980 s(-1), respectively. In vitro cytotoxicity assays with 3677 human melanoma cells demonstrated that C-Mel was 40-fold less toxic than melphalan and was activated in an immunologically specific manner by L49-sFv-bL, a recombinant fusion protein that binds to the melanotransferrin antigen on melanomas and on some carcinomas. L49-sFv-bL in combination with C-Mel led to regressions and cures of established subcutaneous 3677 tumors in nude mice. The effects were significantly greater than those of melphalan, which did not result in any long-term regressions in this tumor model. The therapeutic effects were comparable to those obtained in mice treated with the previously described L49-sFv-bL/7-(4-carboxybutanamido)-cephalosporin mustard (CCM) combination. However, C-Mel may be more attractive than CCM for clinical development since the released drug is clinically approved.
Asunto(s)
Antineoplásicos Alquilantes , Cefalosporinas/síntesis química , Melfalán/análogos & derivados , Melfalán/química , Profármacos/síntesis química , Animales , Anticuerpos Monoclonales/genética , Cefalosporinas/metabolismo , Cefalosporinas/uso terapéutico , Humanos , Inmunotoxinas/uso terapéutico , Melanoma/tratamiento farmacológico , Melanoma/inmunología , Melfalán/síntesis química , Melfalán/metabolismo , Melfalán/uso terapéutico , Ratones , Ratones Desnudos , Proteínas Recombinantes de Fusión , Células Tumorales Cultivadas , beta-Lactamasas/genética , beta-Lactamasas/metabolismoRESUMEN
L6 is an IgG2a murine monoclonal antibody which we have demonstrated binds well to HT29 human colon carcinoma cells by flow cytometry, whole cell ELISA, and mixed hemadsorption. In vitro cytotoxicity studies revealed that the monoclonal antibody L6-cytosine deaminase (L6-CD) immunoconjugate plus the nontoxic prodrug, 5-fluorocytosine (5-FC), is equivalent to 5-fluorouracil (5-FU) in its ability to kill HT29 cells. Human alpha-interferon (A/D) was able to enhance this cytotoxic effect. The I.C.50's revealed that very small amounts of L6-CD are needed for this cytotoxic effect (approximately, 5 pg/ml resulted in 50% viability). The limiting factor was the amount of 5-FC employed with L6-CD (3 microM yielded 50% cell viability). alpha-Interferon (A/D) lowered the requirement of 5-FC to 1 microM to achieve 50% cell lethality. In vivo biodistribution experiments indicated that 1 microgram of L6-CD is nonspecifically taken up by the liver and spleen and cleared rapidly from the blood. Significant localization of L6-CD to HT29 tumors occurred only when 99 micrograms of unlabeled L6-CD was added to 1 microgram of 125I-labeled immunoconjugate injected intravenously. Further augmentation of tumor/blood ratios without reduction in percent injected dose per gram of tumor was possible with the intravenous injection of 100 micrograms of anti-idiotypic monoclonal antibody 13B, 24 hours after L6-CD, which bound unreacted L6-CD and cleared it from the blood. The addition of 100,000 U of alpha-interferon (A/D) given intraperitoneally every day increased the clearance of L6-CD by the liver and spleen, but impaired tumor localization (percent injected dose per gram). These studies demonstrated that in vivo localization of the L6-CD conjugate to HT29 tumors could be optimized by injecting excess L6-CD followed by an equal amount of L6 anti-idiotype mAb 13B, 24 hours after L6-CD.
Asunto(s)
Adenosina Desaminasa/farmacocinética , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Monoclonales/toxicidad , Neoplasias del Colon/tratamiento farmacológico , Inmunotoxinas/farmacocinética , Inmunotoxinas/toxicidad , Interferón Tipo I/farmacología , Animales , Anticuerpos Antiidiotipos/sangre , Supervivencia Celular/efectos de los fármacos , Flucitosina/farmacocinética , Flucitosina/toxicidad , Hemabsorción , Humanos , Hígado/metabolismo , Ratones , Ratones Desnudos , Proteínas Recombinantes , Bazo/metabolismo , Distribución Tisular , Células Tumorales CultivadasRESUMEN
Antitumor immunotoxins were formed by covalently attaching the ribosome-inactivating protein ricin A chain (RA) to the antitumor antibodies BR96 and L6. In vitro cytotoxicity assays established that BR96-RA was cytotoxic to H2987 human lung adenocarcinoma cells (IC50 = 6 nM), while L6-RA exhibited very low levels of cytotoxic activity (18% cell kill at 67 nM). The virulence factor from the intracellular pathogen Listeria monocytogenes, listeriolysin O (LLO), was able to potentiate the cytotoxicity of BR96-RA and L6-RA by 120- and > 1340-fold, respectively, resulting in IC50 values of approximately 50 pM. LLO also potentiated the cytotoxicity of the peptide anticancer drug bleomycin by a factor of > 2500 but had no effect on the cytotoxic activities of the anticancer drugs cytarabine and etoposide phosphate. In addition, LLO did not potentiate the cytotoxic activity of unconjugated ricin A chain or L6-RA on H2987 cells that were saturated with L6 prior to conjugate treatment. These results are attributed to LLO-induced alteration of the intracellular trafficking of molecules that are incorporated into acidic vesicles.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Toxinas Bacterianas , Neoplasias Pulmonares/tratamiento farmacológico , Antibióticos Antineoplásicos/administración & dosificación , Proteínas Bacterianas/administración & dosificación , Bleomicina/administración & dosificación , Sinergismo Farmacológico , Proteínas de Choque Térmico/administración & dosificación , Proteínas Hemolisinas/administración & dosificación , Humanos , Inmunotoxinas/administración & dosificación , Ricina/administración & dosificación , Células Tumorales CultivadasRESUMEN
The syntheses of two cephalosporin derivatives 2 and 3 of mitomycin C (1) containing 7-phenylacetamido and 7-delta-carboxybutanamido side chains, respectively, are described. These compounds were prepared for evaluation as cephalosporin prodrugs capable of being activated by mAb-beta-lactamase conjugates. In vitro cytotoxicity assays performed on H2987 lung adenocarcinoma and clone 62 melanoma cell lines indicated that compound 2 was comparable in cytotoxicity to the parent drug. In an effort to improve upon the cytotoxic differential of 2, an alternative prodrug 3 containing a polar carboxyl group in the side chain of the cephalosporin moiety was prepared. Compound 3 consistently behaved as a prodrug and was approximately 40- and 10-fold less toxic than 1 toward H2987 and clone 62, respectively. Determination of kinetic constants for hydrolysis by beta-lactamase from Enterobacter cloacae P99 indicated kcat values of 476 +/- 170 and 248 +/- 15.1 s-1 for 2 and 3, respectively. The kcat/Km ratios for 2 and 3 were found to be approximately 9.7 and 2.1 microM/s, respectively. Comparison of these kcat/Km values with those obtained for similar cephalosporin derivatives of other antitumor agents demonstrated that compounds with delta-carboxybutanamido side chains generally have slightly diminished efficiency of enzymatic hydrolysis compared to the corresponding 7-phenylacetamido analog. It was also demonstrated that the less toxic prodrug 3 was activated in an immunologically specific manner by L6-F(ab')-beta-lactamase and 96.5-F(ab')-beta-lactamase conjugates, selective for H2987 and clone 62 cells, respectively.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Cefalosporinas/química , Mitomicinas , beta-Lactamasas/inmunología , Cefalosporinas/inmunología , Humanos , Sustancias Macromoleculares , Mitomicina/inmunología , Mitomicina/metabolismo , Modelos Químicos , Profármacos/síntesis química , Células Tumorales CultivadasRESUMEN
The L49 (IgG1) monoclonal antibody binds to p97 (melanotransferrin), a tumor-selective antigen that is expressed on human melanomas and carcinomas. A recombinant fusion protein, L49-sFv-bL, that contains the antibody binding regions of L49 fused to the Enterobacter cloacae r2-1 beta-lactamase (bL) was constructed, expressed, and purified to homogeneity in an Escherichia coli soluble expression system. The variable regions of L49 were cloned by reverse transcription-polymerase chain reaction from L49 hybridoma mRNA using signal sequence and constant region primers. Construction of the gene encoding L49-sFv-bL was accomplished by hybridization insertion of VH, VL, and sFv linker sequences onto a pET phagemid template containing the bL gene fused to the pelB leader sequence. Optimal soluble expression of L49-sFv-bL in E. coli was found to take place at 23 degrees C with 50 microM isopropyl beta-D-thiogalactopyranoside induction and the use of the nonionic detergent Nonidet P-40 for isolation from the bacteria. Construction and expression of a soluble form of the p97 antigen in Chinese hamster ovary cells allowed affinity-based methods for analysis and purification of the fusion protein. Surface plasmon resonance, fluorescent activated cell sorting, and Michaelis-Menten kinetic analyses showed that L49-sFv-bL retained the antigen binding capability of monovalent L49 as well as the enzymatic activity of bL. In vitro experiments demonstrated that L49-sFv-bL bound to 3677 melanoma cells expressing the p97 antigen and effected the activation of 7-(4-carboxybutanamido)cephalosporin mustard (CCM), a cephalosporin nitrogen mustard prodrug. On the basis of these results, L49-sFv-bL was injected into nude mice with subcutaneous 3677 tumors, and localization was determined by measuring bL activity. Tumor to blood conjugate ratios of 13 and 150 were obtained 4 and 48 h post conjugate administration, respectively, and the tumor to liver, spleen, and kidney ratios were even higher. A chemically produced L49-Fab'-bL conjugate yielded a much lower tumor to blood ratio (5.6 at 72 h post administration) than L49-sFv-bL. Therapy experiments established that well-tolerated doses of L49-sFv-bL/CCM combinations resulted in cures of 3677 tumors in nude mice. The favorable pharmacokinetic properties of L49-sFv-bL allowed prodrug treatment to be initiated 12 h after the conjugate was administered. Thus, L49-sFv-bL appears to have promising characteristics for site-selective anticancer prodrug activation.
Asunto(s)
Antineoplásicos/farmacocinética , Inmunoglobulina G/genética , Profármacos/farmacocinética , beta-Lactamasas/genética , Animales , Biotransformación , Células CHO , Clonación Molecular , Cricetinae , Enterobacter cloacae/genética , Escherichia coli/genética , Humanos , Inmunoglobulina G/farmacología , Inmunoglobulina G/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias Experimentales/terapia , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes de Fusión/uso terapéutico , Células Tumorales Cultivadas , beta-Lactamasas/farmacología , beta-Lactamasas/uso terapéuticoRESUMEN
The interaction of organometallic compounds with biological systems, generally called bioorganometallic chemistry, is receiving increasing interest. We present the first part of our studies concerning the biological activity of organometallic compounds. Several alkyne-cobalt carbonyl complexes inhibited the growth of human melanoma and lung carcinoma cell lines. They are more active than uncomplexed dicobalt octacarbonyl, cobalt chloride, or the free ligand. A significant difference in potency towards the lung carcinoma cell line was observed among the cobalt complexes, indicating that the complexed ligand may influence cytotoxic activity. These results suggest that further exploratory work with such cobalt-alkyne complexes is warranted.
Asunto(s)
Antineoplásicos/síntesis química , Compuestos Organometálicos/síntesis química , Adenocarcinoma , Antineoplásicos/química , Antineoplásicos/toxicidad , Supervivencia Celular/efectos de los fármacos , Cobalto/toxicidad , Humanos , Neoplasias Pulmonares , Melanoma , Compuestos Organometálicos/química , Compuestos Organometálicos/toxicidad , Células Tumorales CultivadasRESUMEN
Interaction of the CD80 (B7-1) and CD86 (B7-2) molecules on antigen presenting cells with the receptors CD28 and CTLA-4 on T cells generates signals important in the regulation of immune responses. Because this receptor system involves multiple receptor-ligand interactions, determining the function for individual receptors has been difficult. One approach is the use of antibodies and their derivatives with singular specificity as substitute ligands to explore the activities of these molecules. We have constructed recombinant mono- and bi-specific sFv molecules specific for the CD28 receptor that are capable of binding and generating costimulatory signals to activate T cells. We demonstrate that these soluble molecules are capable of higher levels of costimulation than soluble CD80Ig at equivalent concentrations. We also constructed artificial adhesion receptors on the cell surface using two different CD28-specific sFvIgs fused to the CD80 cytoplasmic and transmembrane domains. In this report, we compared costimulation by a soluble bispecific (alpha CD28-alpha L6) single chain sFvIg fusion protein to that generated by L6 antigen positive (L6+) H3347 tumor cells transduced with cell surface expressed forms of alpha CD28 sFv's. We show that the bispecific protein can target potent CD28 costimulatory activity to L6+ tumor cells in vitro. We also show that transfection of the cell surface forms of the two different CD28 sFvIgs into H3347 tumor cells allows them to generate significant costimulatory signals to activated T cells. Finally, we demonstrate that tumor cell presentation of either the soluble bispecific or transduced cell surface sFv generate similar costimulatory effects resulting in T cell activation.
Asunto(s)
Antígenos de Superficie/inmunología , Antígenos CD28/inmunología , Fragmentos de Inmunoglobulinas/inmunología , Proteínas de Neoplasias/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Antígenos de Superficie/genética , Antígeno B7-1/inmunología , Células CHO , Células COS , Clonación Molecular , Cricetinae , Reactivos de Enlaces Cruzados/farmacología , ADN , Expresión Génica , Humanos , Fragmentos de Inmunoglobulinas/genética , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Mitógenos , Datos de Secuencia Molecular , Proteínas de Neoplasias/genética , Fitohemaglutininas/farmacología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Células Tumorales CultivadasRESUMEN
Paclitaxel-2-ethylcarbonate (PC) is a prototype for a family of paclitaxel prodrugs that have significant levels of antitumor activities in rodent models for human cancer. In this study, an enzyme responsible for the conversion of PC to paclitaxel was purified from rat serum. N-terminal amino acid sequence analysis indicated that the isolated enzyme was rat serum carboxylesterase. This enzyme was shown to significantly enhance the cytotoxic activities of both PC and 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11), a water-soluble analogue of camptothecin, on lung carcinoma and melanoma cell lines. Rat serum carboxylesterase may have applications for the site-specific delivery of anticancer drugs to tumor masses.
Asunto(s)
Antineoplásicos Fitogénicos/metabolismo , Camptotecina/metabolismo , Hidrolasas de Éster Carboxílico/fisiología , Paclitaxel/metabolismo , Profármacos/metabolismo , Animales , Biotransformación , Carboxilesterasa , Hidrolasas de Éster Carboxílico/sangre , Humanos , Ratones , RatasRESUMEN
A library of Enterobacter cloacae P99 beta-lactamase mutants was produced to investigate the importance of residues 286-290 for substrate binding and catalysis and to characterize mutants with altered specificities and activities for various 3'-substituted cephalosporins. This region of the enzyme is a component of the active site that has not been implicated as participating in the catalytic mechanism but, based on molecular modeling, should contact the 3' substituents of cephalosporins. Random mutagenesis was carried out within an M13 phage vector by hybridization mutagenesis, and the phage library could be highly enriched for active beta-lactamase genes by incubation of infected bacteria with beta-lactam antibiotics. The mutants were characterized by Michaelis-Menten kinetic analyses with several cephalosporin substrates and spanned a 25-fold range of k(cat), 24-fold range of K(m), and 6-fold range of k(cat)/K(m) values. All five amino acid positions were found to be permissive to substitution, but the active mutant proteins carried substitutions that likely maintained the structure of the region. Serine 287 was the least permissive to change, requiring small, uncharged residues for retention of catalytic activity. The variation of Michaelis-Menten kinetic parameters observed in these enzymes was shown to be significant in the context of in vitro cytotoxicity assays with the cephalosporin-doxorubicin prodrug C-Dox and is suitable for experiments to probe the relationship between enzyme kinetics and efficacy in enzyme-prodrug approaches to targeted therapy.
Asunto(s)
Bacteriófago M13/genética , Enterobacter cloacae/enzimología , Vectores Genéticos , beta-Lactamasas/metabolismo , Secuencia de Bases , Catálisis , Supervivencia Celular , Cartilla de ADN , Cinética , Datos de Secuencia Molecular , Mutagénesis , Especificidad por Sustrato , Células Tumorales Cultivadas , beta-Lactamasas/genéticaRESUMEN
Cephalosporin doxorubicin (C-Dox) and 7-(4-carboxybutanamido)-cephalosporin mustard (CCM) are prodrugs that are catalytically converted by Enterobacter cloacae beta-lactamase (bL) to the active anticancer agents doxorubicin and phenylenediamine mustard, respectively. Both prodrugs were less cytotoxic to the 3677 human melanoma line than their respective drugs and were activated in an immunologically specific manner by 96.5-bL, a mAb-bL conjugate that binds to 3677 cell surface antigens. Similar results were obtained using the CCM prodrug on SK-MEL 28 human melanoma cells. Experiments in mice with established s.c. 3677 tumors demonstrated that although no tumors were cured in mice receiving the 96.5-bL/C-Dox combination, the activities were greater than those obtained from systemic doxorubicin treatment or from administration of the nonbinding conjugate P1.17-bL in combination with C-Dox. In contrast, when CCM was used as a prodrug, cures of established 3677 tumors were obtained in 80% of the 96.5-bL treated animals. This combination was also able to induce regressions of large 3677 tumor masses (800 mm3) without any apparent toxic side effects. We conclude that 96.5-bL in combination with C-Dox or CCM has greater antitumor activity than systemic treatment with the corresponding drugs and that CCM is a more effective prodrug than C-Dox for treating human 3677 melanoma xenografts.
Asunto(s)
Doxorrubicina/administración & dosificación , Melanoma/tratamiento farmacológico , Compuestos de Mostaza Nitrogenada/administración & dosificación , Profármacos/administración & dosificación , beta-Lactamasas/administración & dosificación , Animales , Anticuerpos Monoclonales/uso terapéutico , Antígenos de Neoplasias , Cefalosporinas , Femenino , Humanos , Inmunoconjugados , Antígenos Específicos del Melanoma , Ratones , Ratones Desnudos , Proteínas de Neoplasias/inmunología , Trasplante de NeoplasiasRESUMEN
7-Aminocephalosporin doxorubicin (AC-Dox) was condensed with monomethoxypoly(ethylene glycol)-propionic acid N-hydroxysuccinimide ester (5 kDa) or with a branched form of poly(ethylene glycol)-propionic acid N-hydroxysuccinimide ester (10 kDa), forming M-PEG-AC-Dox and B-PEG-AC-Dox, respectively. These polymer drug derivatives were designed such that doxorubicin would be released upon Enterobacter cloacae beta-lactamase (bL)-catalyzed hydrolysis. Both M-PEG-AC-Dox (IC50 = 80 microM) and B-PEG-AC-Dox (IC50 = 8 microM) were less toxic to H2981 human lung adenocarcinoma cells than doxorubicin (IC50 = 0.1-0.2 microM) and could be activated in an immunologically specific manner by L6-bL, a monoclonal antibody-bL conjugate that bound to H2981 cell surface antigens. In addition, the polymers were relatively stable in mouse plasma (< 26% hydrolysis after 24 h at 37 degrees C) and were less toxic to mice (maximum tolerated dose > 52 mumol/kg) than doxorubicin (maximum tolerated dose = 13.8 mumol/kg). Pharmacokientic studies were performed in mice bearing subcutaneous 3677 melanoma tumors. B-PEG-AC-Dox cleared from the blood more slowly than M-PEG-AC-Dox and was retained to a 2.1-fold greater extent in human 3677 melanoma tumor xenografts over a 4 h period. The intratumoral concentrations of both polymers far exceeded that of doxorubicin. Thus, the PEG-AC-Dox polymers offer the possibility of generating large intratumoral doxorubicin concentrations owing to their reduced toxicities, the amounts that accumulate in tumors, and the fact that doxorubicin is released upon beta-lactam ring hydrolysis.
Asunto(s)
Cefalosporinas/síntesis química , Cefalosporinas/toxicidad , Doxorrubicina/análogos & derivados , Doxorrubicina/farmacocinética , Inmunotoxinas/farmacocinética , Inmunotoxinas/toxicidad , Melanoma/tratamiento farmacológico , Polietilenglicoles , Polietilenglicoles/síntesis química , Polietilenglicoles/toxicidad , beta-Lactamasas/metabolismo , Adenocarcinoma , Animales , Anticuerpos Monoclonales , Supervivencia Celular/efectos de los fármacos , Cefalosporinas/farmacocinética , Doxorrubicina/síntesis química , Doxorrubicina/toxicidad , Enterobacter cloacae/enzimología , Femenino , Humanos , Inmunotoxinas/uso terapéutico , Neoplasias Pulmonares , Ratones , Ratones Desnudos , Estructura Molecular , Polietilenglicoles/farmacocinética , Relación Estructura-Actividad , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
A cephalosporin derivative of doxorubicin (C-Dox) was evaluated as a prodrug for activation by mAb conjugates of the beta-lactamase from Enterobacter cloacae P99 (beta L; EC 3.5.2.6). The conjugates consisted of beta L and the F(ab') fragments of either of the mAbs L6, P1.17, or 96.5. L6 binds to antigens on a variety of carcinomas, including the two lung adenocarcinoma cell lines H2981 and H2987 used in this study. 96.5 binds to the melanoma-associated antigen p97, and P1.17 was used for the control conjugate. C-Dox was found to be less cytotoxic to three different tumor cell lines in vitro compared to the parent drug doxorubicin (Dox). Immunospecific activation took place when the cells were pretreated with beta L conjugates that could bind to antigens on the tumor cells. In vivo toxicity and pharmacokinetics studies in athymic female nu/nu mice revealed that C-Dox was at least 7-fold less toxic than Dox (on a molar basis), despite the fact that a > or = 320-fold greater area-under-the-curve (blood concentration versus time) of C-Dox compared to Dox was obtained 0-2 h after administration of the two agents. Pharmacokinetic studies at maximum tolerated doses in mice bearing xenografts of either H2981 or H2987 revealed that the intratumoral levels of Dox after treatment with L6-beta L in combination with C-Dox were higher than were obtained by either systemic treatment with Dox or a combination of P1.17-beta L and C-Dox. This finding suggested that the conversion of C-Dox to Dox was tumor specific and dependent on the presence of the targeted antigen. Furthermore, the best antitumor activity against both H2981 and H2987 tumors was obtained by treatment with L6-beta L and C-Dox compared to P1.17-beta L and C-Dox or Dox alone. Thus, higher levels of Dox corresponded to greater therapeutic effects in both of the tumor models studied.