RESUMEN

Clostridium difficile is the bacterial pathogen identified as the cause of pseudomembranous colitis and is principally responsible for nosocomial antibiotic-associated diarrhea and colitis. The pathologic findings associated with this infection are believed to be caused by two large (approximately 300-kDa) exotoxins, toxins A and B. Because of the mucosal nature of this infection, vaccination strategies aimed at providing prophylactic or therapeutic immune protection have included immunization by mucosal routes. Using the hamster model of C. difficile infection, we examined the protective efficacy of inactivated toxin (toxoid) vaccine formulations prepared as either culture filtrate or partially purified toxoid. We compared combination parenteral and mucosal vaccination regimens involving intranasal, intragastric, or rectal routes of immunization and found that rectal immunization in conjunction with intramuscular (i.m.) vaccination provided full protection of hamsters from death and diarrhea while the other mucosal routes did not. Protection was associated with high levels of toxin-neutralizing antibodies in serum. The requirement for adjuvants for protection was assessed by using sequential i.m. and rectal or i.m. vaccination regimens. Unexpectedly, i.m. immunization without adjuvant conferred the highest protection from death and diarrhea; this regimen elicited the highest serum anti-toxin B titers as well as toxin B neutralizing titers. Passive transfer of mouse antitoxin antibodies protected hamsters in a dose-dependent manner, demonstrating the principal role of circulating antitoxin antibodies in immunity from this toxin-mediated mucosal disease. These results suggest that prophylactic parenteral vaccination or intravenous immunotherapy could provide protection from C. difficile disease in humans.

Asunto(s)

Antitoxinas/inmunología , Clostridioides difficile/inmunología , Enterocolitis Seudomembranosa/prevención & control , Animales , Anticuerpos Antibacterianos/inmunología , Antitoxinas/sangre , Vacunas Bacterianas/inmunología , Ciego , Cricetinae , Diarrea/inmunología , Diarrea/prevención & control , Modelos Animales de Enfermedad , Enterocolitis Seudomembranosa/inmunología , Femenino , Inmunización Pasiva , Infusiones Parenterales , Mesocricetus , Ratones , Membrana Mucosa , Pruebas de Neutralización , Vacunación

RESUMEN

Radioimmunotherapy (RIT) is currently limited by toxicity to normal tissues as a result of prolonged circulating radioantibody in the blood. In this study, the use of a clearing antibody was investigated (second antibody) in an attempt to reduce blood background levels of [90Y]A5B7 immunoglobulin G (IgG) activity, and, therefore, improve the therapeutic tumour-blood ratio in nude mice bearing human colorectal tumour xenografts. The second antibody was raised against the 12N4 macrocycle group used for chelation of 90Y, and is, thus, applicable to any anti-tumour antibody labelled with this methodology. Second antibody was administered 18, 24 or 48 h after radiolabelled antibody injection and produced up to a tenfold reduction in blood levels and a tenfold improvement in tumour-blood ratios. This has the advantage of reducing the risk of myelotoxicity caused by prolonged retention of activity in the blood. For all normal tissues, there was a similar or slightly lower uptake of [90Y]IgG with second antibody clearance, apart from a transient rise in liver activity due to complexes of primary and secondary antibody clearing via the liver. As a result of clearance of [90Y]IgG from the blood pool, there was an associated fall in the amount of antibody at the tumour site (up to 3.3-fold) at later time points for mice injected with second antibody. However, despite this, tumour-blood ratios remained superior to the control group at these later time points. Estimated dosimetry evaluation revealed that total dose to normal tissues, blood and tumour was lower than for the non-clearance group. Surprisingly, however, there was little improvement in total estimated tumour-blood dose ratio over the time period studied. This was probably because the majority of the dose was delivered to both the blood and tumour within the first 24 h after administration of [90Y]IgG, so that giving the clearing agent after this time did not produce a large difference in total estimated dose. The anti-macrocycle second antibody proved to be a successful clearing agent and could potentially be applied to any anti-tumour antibody coupled with the 12N4 macrocycle. In the light of the estimated dosimetry results described here, it would probably be most useful given at earlier time points (i.e. before 18 h after injection of primary antibody) to produce an improved tumour-blood ratio of total dose. Development of this strategy may allow higher levels of activity to be administered for RIT, and repeated dosing regimens.

Asunto(s)

Anticuerpos Antineoplásicos/metabolismo , Compuestos Heterocíclicos/inmunología , Radioinmunoterapia , Radioisótopos de Itrio/farmacocinética , Animales , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/radioterapia , Quimioterapia Combinada , Humanos , Inmunoglobulina G/análisis , Inmunoglobulina G/metabolismo , Ratones , Trasplante de Neoplasias , Distribución Tisular

RESUMEN

The nasal cavity of a rodent is lined by an epithelium organized into distinct regional domains responsible for specific physiological functions. Aggregates of nasal lymphoid tissue (NALT) located at the base of the nasal cavity are believed to be sites of induction of mucosal immune responses to airborne antigens. The epithelium overlying NALT contains M cells which are specialized for the transcytosis of immunogens, as demonstrated in other mucosal tissues. We hypothesized that NALT M cells are characterized by distinct glycoconjugate receptors which influence antigen uptake and immune responses to transcytosed antigens. To identify glycoconjugates that may distinguish NALT M cells from other cells of the respiratory epithelium (RE), we performed lectin histochemistry on sections of the hamster nasal cavity with a panel of lectins. Many classes of glycoconjugates were found on epithelial cells in this region. While most lectins bound to sites on both the RE and M cells, probes capable of recognizing alpha-linked galactose were found to label the follicle-associated epithelium (FAE) almost exclusively. By morphological criteria, the FAE contains >90% M cells. To determine if apical glycoconjugates on M cells were accessible from the nasal cavity, an M-cell-selective lectin and a control lectin in parallel were administered intranasally to hamsters. The M-cell-selective lectin was found to specifically target the FAE, while the control lectin did not. Lectin bound to M cells in vivo was efficiently endocytosed, consistent with the role of M cells in antigen transport. Intranasal immunization with lectin-test antigen conjugates without adjuvant stimulated induction of specific serum immunoglobulin G, whereas antigen alone or admixed with lectin did not. The selective recognition of NALT M cells by a lectin in vivo provides a model for microbial adhesin-host cell receptor interactions on M cells and the targeted delivery of immunogens to NALT following intranasal administration.

Asunto(s)

Antígenos/administración & dosificación , Glicoconjugados/aislamiento & purificación , Lectinas/administración & dosificación , Tejido Linfoide/inmunología , Mucosa Nasal/inmunología , Vacunación/métodos , Administración Intranasal , Animales , Antígenos/inmunología , Cricetinae , Femenino , Galactósidos/aislamiento & purificación , Galactósidos/metabolismo , Glicoconjugados/metabolismo , Histocitoquímica , Lectinas/metabolismo , Tejido Linfoide/química , Tejido Linfoide/citología , Mesocricetus , Mucosa Nasal/química , Mucosa Nasal/citología , Unión Proteica , Aglutinina del Germen de Trigo-Peroxidasa de Rábano Silvestre Conjugada/administración & dosificación , Aglutinina del Germen de Trigo-Peroxidasa de Rábano Silvestre Conjugada/inmunología

RESUMEN

The enzyme carboxypeptidase G2 (CPG2) can be targeted to tumors by antibodies and used to activate prodrugs in a treatment called antibody-directed enzyme prodrug therapy (ADEPT). Different doses of CPG2 conjugated to the anti-CEA antibody A5B7 were administered i.v. to nude mice bearing the LS174T human colon adenocarcinoma xenograft, and the biodistribution of conjugate activity 48 and 72 h later was determined using a novel high-performance liquid chromatography (HPLC) method. Conjugate doses of 2,500 and 625 U/kg gave tumor enzyme levels of 0.5-0.6 U/g. Lower doses of 300 and 150 U/kg gave tumor enzyme levels of 0.1-0.3 U/g. Intriguingly, the best tumor:blood ratio of conjugate activity at both 48 and 72 h was achieved after administration of the 625-U/kg dose, not the 2,500-U/kg dose. After 48 h this ratio was 3.8, whereas after 72 h the value was 5.5. This conjugate dose also gave the greatest tumor:tissue ratios in all other tissues examined. After 72 h the tumor:colon ratio was 105, whereas the tumor:kidney ratio was 36. In ADEPT, to obtain maximal tumor damage to LS174T xenografts in nude mice with minimal systemic toxicity using the A5B7-CPG2 conjugate, prodrug should therefore be administered at least 72 h after a conjugate dose of 625 U/kg.

Asunto(s)

Adenocarcinoma/metabolismo , Anticuerpos Monoclonales/farmacocinética , Neoplasias del Colon/metabolismo , Glutamatos/uso terapéutico , Inmunoconjugados/farmacocinética , Compuestos de Mostaza Nitrogenada/uso terapéutico , Profármacos/uso terapéutico , gamma-Glutamil Hidrolasa/farmacocinética , Adenocarcinoma/tratamiento farmacológico , Animales , Antineoplásicos/uso terapéutico , Antígeno Carcinoembrionario/inmunología , Cromatografía Líquida de Alta Presión , Neoplasias del Colon/tratamiento farmacológico , Femenino , Ratones , Ratones Desnudos , Distribución Tisular , Trasplante Heterólogo

RESUMEN

Radioimmunotherapy (RIT) does not readily eradicate common solid tumors and therefore requires augmentation by complementary therapies that do not increase normal tissue damage. We have examined the efficacy of RIT combined with 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a drug which induces immunomodulation and cytokine production and preferentially reduces tumor blood flow, using a colorectal xenograft model in nude mice. Although an optimal i.p. dose (27.5 mg/kg) of drug alone induced massive hemorrhagic necrosis of all but a thin peripheral rim of viable tumor cells, survival was unaffected. However, when combined with i.v. 18.5 MBq 131I-labeled anti-carcinoembryonic antigen IgG, DMXAA significantly potentiated the RIT without increased toxicity, with five of six mice showing complete cures. Scheduling was critical because the antibody must be allowed to reach maximum tumor accumulation before initiation of drug-induced blood flow inhibition. Subsequently, the antibody was retained preferentially in the tumor, reaching approximately twice control levels by 5 days after drug delivery. In combined studies, the drug had a narrow therapeutic window, 30 mg/kg being toxic to two of six mice, whereas 20 mg/kg were ineffective. However, the addition of a second vasoactive agent, serotonin, to RIT plus 20 mg/kg DMXAA enhanced therapy without increasing systemic toxicity. Tumor histology and phosphor image plate analysis reflected these results. When given without RIT, the two drugs combined, although not alone, also significantly inhibited tumor growth. Drug-induced tumor necrosis and tumor retention of radioantibody may both contribute to the enhanced RIT produced by this combined complementary therapy.

Asunto(s)

Neoplasias Colorrectales/terapia , Radioinmunoterapia/métodos , Xantonas , Animales , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Antineoplásicos/metabolismo , Anticuerpos Antineoplásicos/uso terapéutico , Antígeno Carcinoembrionario/inmunología , Neoplasias Colorrectales/irrigación sanguínea , Terapia Combinada , Flavonoides/administración & dosificación , Humanos , Ratones , Trasplante de Neoplasias , Neoplasias Experimentales/irrigación sanguínea , Flujo Sanguíneo Regional/efectos de los fármacos , Serotonina/farmacología , Distribución Tisular/efectos de los fármacos , Trasplante Heterólogo , Xantenos/administración & dosificación

RESUMEN

Polyethylene glycol modification of the antibody--enzyme conjugate, F(ab')2-A5B7-CPG2, extends its duration in the circulation of nude mice bearing human colonic cancer xenografts (LS174T). Increased concentration of modified conjugate is achieved in the tumour, but residual non-specific enzyme concentrations in normal tissue and blood demonstrate the fundamental requirement to remove or inactivate non-specifically held enzyme in this system.

Asunto(s)

Neoplasias del Colon/metabolismo , gamma-Glutamil Hidrolasa/farmacocinética , Animales , Anticuerpos Monoclonales , Antígeno Carcinoembrionario/inmunología , Neoplasias del Colon/sangre , Humanos , Fragmentos Fab de Inmunoglobulinas , Radioisótopos de Yodo , Ratones , Ratones Desnudos , Polietilenglicoles , Técnica de Dilución de Radioisótopos , Análisis de Regresión , Factores de Tiempo , Distribución Tisular , Trasplante Heterólogo

RESUMEN

UNLABELLED: Single-chain Fv (scFv) antibody fragments have potential for clinical imaging because of their rapid tumor penetration and high tumor-to-tissue ratios at early time points. ScFvs clear rapidly from the circulation so radiolabels such as 99mTc which have short half-lives are desirable, but the free thiol groups necessary for labeling with 99mTc are not normally found on these molecules. METHODS: We constructed a vector which enabled a free cysteine to be linked to the C-terminus of scFvs. MFE-23, a scFv directed against carcinoembryonic antigen (CEA), was cloned into this vector and cys-tagged MFE-23 was labeled with 99mTc using a D-glucarate transfer method. RESULTS: The radiolabeled product was stable in vivo and in vitro and showed favorable tumor-to-blood ratios in vivo at early time points (4:1 at 24 hr and 8:1 at 48 hr), although high kidney levels were also detected. CONCLUSION: Our study demonstrates an effective method to enable scFvs radiolabeling with 99mTc and also shows the potential of using a 99mTc-labeled scFv for clinical imaging studies.

Asunto(s)

Fragmentos de Inmunoglobulinas , Radioinmunodetección , Tecnecio , Adenocarcinoma/diagnóstico por imagen , Animales , Neoplasias del Colon/diagnóstico por imagen , Humanos , Marcaje Isotópico , Ratones , Trasplante de Neoplasias , Distribución Tisular , Trasplante Heterólogo , Células Tumorales Cultivadas

RESUMEN

Effective radioimmunotherapy is limited by slow antibody clearance from the circulation, which results in low tumour to blood ratios and restricts the dose of radiolabelled anti-tumour antibody that can be safely administrated. Avidin and streptavidin clearing agents have been shown to effectively complex and clear radioactive biotinylated antibodies from the circulation, but their immunogenicity may limit their repeated use. We have investigated whether polyethylene glycol (PEG) modification can reduce the immunogenicity of our galactosylated streptavidin (gal-streptavidin) clearing agent without altering its effectiveness as a clearing agent. The immune response evoked in mice after intraperitoneal infection of 30 micrograms of gal-streptavidin was decreased after PEG modification, as shown by lower antibody titres and a reduction in the number of mice that elicited an anti-gal-streptavidin response. The effect of PEG-modified gal-streptavidin on the blood clearance and tumour localisation of a 125I-labelled biotinylated anti-CEA was investigated in the LS174T human colon carcinoma xenograft in nude mice. Although PEG modified gal-streptavidin bound the [125I]biotinylated antibody in vivo, effective clearance from the circulation was inhibited, resulting in very little reduction in the levels of circulation radioactivity, together with a decrease in the antibody localised to the tumour.

Asunto(s)

Anticuerpos Monoclonales/metabolismo , Anticuerpos Antineoplásicos/metabolismo , Proteínas Bacterianas/farmacología , Polietilenglicoles/farmacología , Animales , Anticuerpos Monoclonales/farmacocinética , Proteínas Bacterianas/inmunología , Biotina , Cromatografía en Gel , Humanos , Tasa de Depuración Metabólica , Ratones , Peso Molecular , Radioinmunoterapia , Estreptavidina

RESUMEN

BACKGROUND: There is considerable interest in the specific targeting of therapeutic agents to cancer cells. Of particular promise is a technique known as Antibody-Directed Enzyme Prodrug Therapy (ADEPT). In this approach an enzyme is targeted to the tumour by its conjugation to a tumour specific-antibody tumour. After allowing sufficient time for the conjugate to localise at the tumour and clear from the circulatory system, a relatively non-toxic prodrug is administered. This prodrug is converted to a highly cytotoxic drug by the action of the targeted enzyme localised at the tumour site. OBJECTIVES: To construct gene fusions between the pseudomonad carboxypeptidase G2 (CPG2) gene and DNA encoding MFE-23 (an anti-carcinoembryonic antigen (CEA) single-chain Fv (scFv) molecule), derived from a phage display library. To overexpress the resultant gene fusions in Escherichia coli, and assess the in vitro and in vivo properties of the purified fusion proteins. STUDY DESIGN: To introduce unique cloning restriction sites into the 5'-end of the CPG2 gene by site-directed mutagenesis to facilitate fusion to the 3'-end of the gene encoding MFE-23 (constructs with or without a flexible (Gly4Ser)3 linker-encoding sequence were designed). To overexpress the resultant gene fusions under transcriptional control of the lac promoter and to direct the fusion proteins produced to the periplasmic space of E. coli through translational coupling to the pelB signal peptide. RESULTS: Biologically active recombinant CPG2::MFE-23 scFv fusion proteins were produced in E. coli and shown to possess enzyme and anti-CEA activity. Affinity chromatography followed by size exclusion gel filtration yielded approximately 0.7-1.4 mg/l from shake flask culture. The fusion protein in which the enzyme and antibody moieties were joined by a linker peptide was shown to be effectively localised in nude mice bearing human colon tumour xenografts, giving favourable tumour to blood ratios. CONCLUSION: MFE-23 scFv serves as an ideal candidate for the antibody arm of a bacterially expressed fusion protein with CPG2. The biological properties of this recombinant protein suggest that it may be employed for tumour specific prodrug activation. However, further assessment of its stability and pharmokinetics is required if genetic fusion is to be considered as an alternative to chemical conjugation.

Asunto(s)

Antígeno Carcinoembrionario/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , gamma-Glutamil Hidrolasa/genética , Animales , Fusión Artificial Génica , Western Blotting , Antígeno Carcinoembrionario/inmunología , Cromatografía de Afinidad , Mapeo Cromosómico , Clonación Molecular , Colifagos/genética , Neoplasias Colorrectales/metabolismo , Escherichia coli/genética , Expresión Génica , Biblioteca de Genes , Glicina/genética , Humanos , Operón Lac , Ratones , Ratones Desnudos , Mutagénesis Sitio-Dirigida , Trasplante de Neoplasias , Polisacárido Liasas/metabolismo , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/inmunología , Serina/genética , Trasplante Heterólogo , gamma-Glutamil Hidrolasa/metabolismo

RESUMEN

Polyethylene glycol (PEG) modification of a chimeric Fab' fragment (F9) of A5B7 (alpha-CEA), using an improved coupling method, increases its specificity for subcutaneous LS174T tumours. PEGylation increased the area under the concentration-time curve (AUC0-144) in all tissues but there were significant differences (variance ratio test, F = 27.95, P < 0.001) between the proportional increases in AUC0-144, with the tumour showing the greatest increase. The increase in AUCtumour from F9 to PEG-F9 was similar to the reported increase from Fab' to F(ab')2 while the increase in AUCblood by PEGylation of F9 was only 21% of the reported increase from Fab' to whole IgG. A two sample t-test showed no significant differences between maximal tumour/tissue ratios for PEG-F9 and F9 while the tumour/tissue ratios for PEG-F9 remained high over a longer period, with tumour levels at least double those for F9. PEG-F9 emerges as a new generation antibody with potential advantages for both radioimmunotherapy and tumour imaging. Since there was a reduction in antigen binding, optimisation of PEGylation might further improve tumour specificity. The latter resulted from complex effects on both the entry into and exit rates from tumour and normal tissues in a tissue-specific fashion.

Asunto(s)

Anticuerpos Monoclonales/farmacocinética , Anticuerpos Antineoplásicos/metabolismo , Especificidad de Anticuerpos/efectos de los fármacos , Antígeno Carcinoembrionario/inmunología , Fragmentos Fab de Inmunoglobulinas/metabolismo , Polietilenglicoles/química , Sulfonas/química , Adenocarcinoma/inmunología , Adenocarcinoma/metabolismo , Animales , Anticuerpos Antineoplásicos/efectos de los fármacos , Sitios de Unión de Anticuerpos , Disponibilidad Biológica , Neoplasias del Colon/inmunología , Neoplasias del Colon/metabolismo , Femenino , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/efectos de los fármacos , Fragmentos Fab de Inmunoglobulinas/inmunología , Modelos Lineales , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Proteínas Recombinantes de Fusión , Sulfonas/farmacología , Distribución Tisular

RESUMEN

The effect of anti-enzyme antibody clearance on prodrug turnover in antibody-directed enzyme prodrug therapy (ADEPT) has been studied. Mice bearing LS174T xenografts were given localising carboxypeptidase G2 (CPG)2 conjugate (AEC) and 19 h later galactosylated anti-CPG2 antibody (SB43-GAL). In regimen I prodrug was injected 5 h after SB43-GAL as previously described. In regimen 2 and 3 a shortened and extended clearance time was used in which prodrug was administered 0.5 h or 53 h after SB43-GAL respectively. Regimen 1 resulted in similar tumour and normal tissue levels of active drug to those of the control in which prodrug was given 72 h after AEC. SB43-GAL therefore accelerated clearance of enzyme allowing early administration of prodrug. In regimen 2, very high active drug levels were found in the liver, showing removal of AEC from the blood followed by reactivation of enzyme and extensive and rapid prodrug turnover. Active drug levels in tumour and blood reached similar peak levels to those of the control. Regimen 3 resulted in lower active drug levels in tissues, consistent with degradation and excretion of enzyme. Regimen 3 also produced the best tumour to normal ratios for active drug. Residual prodrug in tumour was unaffected by SB43-GAL, showing the advantage of galactosylation in minimising inactivation of CPG2 in tumour. By contrast, residual prodrug in blood persisted for longer when SB43-GAL was used. Circulatory clearance of enzyme with SB43-GAL allows prodrug to be administered expediently with reduced toxicity and with the prospect of increasing the dosage.

Asunto(s)

Antineoplásicos/farmacocinética , Inmunoconjugados/sangre , Profármacos/farmacocinética , Profármacos/uso terapéutico , gamma-Glutamil Hidrolasa/farmacocinética , Adenocarcinoma/sangre , Adenocarcinoma/metabolismo , Animales , Anticuerpos Monoclonales/sangre , Anticuerpos Monoclonales/farmacología , Antineoplásicos/sangre , Antineoplásicos/metabolismo , Biotransformación , Antígeno Carcinoembrionario/inmunología , Neoplasias del Colon/sangre , Neoplasias del Colon/metabolismo , Humanos , Inmunoconjugados/metabolismo , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Profármacos/metabolismo , Trasplante Heterólogo , gamma-Glutamil Hidrolasa/metabolismo

RESUMEN

Single-chain antibodies (scFvs) can be derived from a monoclonal antibody (MAb) or produced directly using filamentous phage technology, where antibodies with desired binding and purification characteristics can be readily selected from libraries. To test the hypothesis that the latter approach is more useful, we compared 2 anti-carcinoembryonic antigen (CEA) scFvs produced by these 2 different approaches. Our study showed that, both in the purification process and in the biodistribution pattern, MFE-23, produced by filamentous phage technology, gave favourable results compared to A5-SC, which is derived from the A5B7 MAb. This indicates the value of the filamentous phage approach for obtaining tumour-targeting scFvs.

Asunto(s)

Anticuerpos Antivirales/aislamiento & purificación , Anticuerpos Antivirales/metabolismo , Bacteriófagos/metabolismo , Hibridomas/metabolismo , Región Variable de Inmunoglobulina/biosíntesis , Animales , Anticuerpos Monoclonales/metabolismo , Anticuerpos Antivirales/inmunología , Formación de Anticuerpos , Bacteriófagos/inmunología , Antígeno Carcinoembrionario/inmunología , Cromatografía de Afinidad , Cromatografía en Gel , Clonación Molecular , Neoplasias Colorrectales/diagnóstico por imagen , Neoplasias Colorrectales/metabolismo , Estabilidad de Medicamentos , Humanos , Hibridomas/inmunología , Yodo/farmacocinética , Radioisótopos de Yodo , Punto Isoeléctrico , Ratones , Ratones Desnudos , Cintigrafía , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Distribución Tisular , Trasplante Heterólogo

RESUMEN

Persistence of high levels of radiolabelled antibody in the circulation is a major limitation of radioimmunotherapy. Biotinylation of the radiolabelled anti-tumour antibody followed by administration of streptavidin is known to give much improved tumour to blood ratios as the radioantibody is complexed and subsequently cleared via the reticuloendothelial system, although prolonged splenic uptake is a problem. We have investigated the effect on the clearance pattern and tumour localisation of a 125I-labelled biotinylated anti-CEA antibody (A5B7) after administration of a galactosylated form of streptavidin (gal-streptavidin) in nude mice bearing a human colon carcinoma xenograft. Fifteen minutes to 1 h after gal-streptavidin administration the complexes were cleared via the liver alone (as opposed to liver and spleen after native streptavidin). Twenty-four hours after administration of gal-streptavidin, the tumour to blood ratio for biotinylated A5B7 IgG increased from 2.9 to 13.2 and for biotinylated F(ab')2 fragments an increase from 4.9 to 33.2 was achieved. The reduction in tumour accumulation of F(ab')2 24 h after injection of the clearing agent was less than that seen with intact antibody. Injection of asialofetuin inhibited clearance, confirming that removal of the gal-streptavidin-biotinylated antibody complexes from the blood was via the asialoglycoprotein receptor on liver hepatocytes. Therefore, galactosylation of the streptavidin clearing agent allows rapid removal of radiolabelled biotinylated antibodies via the liver asialoglycoprotein receptor, as opposed to the reticuloendothelial system.

Asunto(s)

Anticuerpos Monoclonales/farmacocinética , Proteínas Bacterianas/farmacología , Antígeno Carcinoembrionario/inmunología , Fragmentos Fab de Inmunoglobulinas/metabolismo , Receptor de Asialoglicoproteína , Asialoglicoproteínas/farmacología , Biotina , Fetuínas , Galactosa , Radioisótopos de Yodo , Tasa de Depuración Metabólica/efectos de los fármacos , Radioinmunodetección , Receptores de Superficie Celular/fisiología , Estreptavidina , Distribución Tisular , alfa-Fetoproteínas/farmacología

RESUMEN

Attachment of poly(ethylene glycol) (PEG) to proteins can greatly alter their pharmacological properties, including extending the plasma half-life and reducing immunogenicity, both of which are potentially beneficial to tumour targeting. IgG, F(ab')2 and Fab' fragments of the anti-CEA antibody A5B7 were chemically modified with PEG (M(r) 5,000), labelled with 125I and their pharmacokinetics compared with the unmodified forms in the LS174T colonic xenograft in nude mice. PEG modification of the intact antibody had little effect on biodistribution, although tumour localisation was slightly reduced. In contrast, similar modification of F(ab')2 and Fab'A5B7 significantly prolonged plasma half-life and increased radioantibody accumulation in the tumour and to a lesser extent in normal tissues, but reduced tissue to blood ratios. Prior to modification, Fab' A5B7 (M(r) 50,000) cleared more rapidly from the circulation than F(ab')2 (M(r) 100,000), but after PEG attachment their biodistributions converged, while the tumour to blood ratios were reduced and resembled that of the intact antibody. The enhanced tumour accumulation, reduced normal tissue to blood ratios and potentially reduced immunogenicity of fragments after PEG attachment may therefore prove superior to either unmodified fragments or intact antibody for antibody-targeted therapy, although the increased plasma half-life may necessitate the use of a clearance mechanism.

Asunto(s)

Adenocarcinoma/inmunología , Anticuerpos Monoclonales/metabolismo , Antígeno Carcinoembrionario/inmunología , Neoplasias del Colon/inmunología , Animales , Anticuerpos Monoclonales/química , Humanos , Fragmentos Fab de Inmunoglobulinas/metabolismo , Técnicas In Vitro , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Polietilenglicoles/química , Distribución Tisular

RESUMEN

Radioimmunotherapy (RIT) is hampered clinically by poor tumour localization of antibody. In order to enhance localization we have investigated the concomitant use of RIT with 2 drugs, flavone-8-acetic acid (FAA) and its analogue 5,6-dimethylxanthenone-4-acetic acid (XAA), which both reduce tumour blood flow and induce immunomodulation. A single i.v. dose of 0.5 mCi (18.5 MBq) intact 131I anti-CEA antibody significantly delayed growth and prolonged survival over that of untreated controls, in an established LS174T colon xenograft model in nude mice. The adjuvant use of a single i.p. dose of either FAA or XAA, given 24 or 48 hr after 131I-A5B7 to allow maximum tumour levels of antibody to be attained before drug-induced blood-flow inhibition, significantly enhanced the RIT. FAA caused entrapment of antibody within the tumour in relation to the time allowed for localization before drug administration. Repeated doses of FAA prolonged tumour growth inhibition but did not enhance the therapy achieved after a single dose. Although both drugs alone induced massive tumour necrosis of all but a thin peripheral rim of viable cells, tumour regrowth was inhibited for a few days only, with no effect on survival. Drug-induced tumour necrosis, immunomodulation and retention of higher doses of 131I-A5B7 within the tumour may contribute to the enhanced RIT produced by this combined therapy.

Asunto(s)

Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Antineoplásicos/administración & dosificación , Neoplasias del Colon/terapia , Flavonoides/uso terapéutico , Animales , Neoplasias del Colon/irrigación sanguínea , Femenino , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Flujo Sanguíneo Regional/efectos de los fármacos , Distribución Tisular/efectos de los fármacos , Células Tumorales Cultivadas

RESUMEN

A recombinant chimeric Fab (rcFab), with Fv derived from the monoclonal A5B7 antibody to carcinoembryonic antigen (CEA), and with human CHI and C kappa was cloned into pUC 19 and expressed in Escherichia coli. rcFab (10 to 12 mg per litre) was produced in bacterial culture fluid, and functional purified rcFab was isolated by affinity chromatography (using antibody to human C kappa) and size-exclusion gel filtration. The rcFab did not show reduced affinity for CEA, and reacted with human colorectal tumours showing a typical anti-CEA pattern by immunocytochemistry; it was also stable after iodination. Biodistribution studies in nude mice bearing human tumour xenografts showed no toxicity and good tumour localization. Therapeutic ratios at early time points were better than those obtained with whole murine antibody. The results demonstrate that bacterially produced anti-CEA Fab is of use for tumour targeting.

Asunto(s)

Adenocarcinoma/metabolismo , Antígeno Carcinoembrionario/inmunología , Neoplasias Colorrectales/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Expresión Génica/genética , Humanos , Regiones Constantes de Inmunoglobulina/biosíntesis , Regiones Constantes de Inmunoglobulina/genética , Regiones Constantes de Inmunoglobulina/metabolismo , Fragmentos de Inmunoglobulinas/biosíntesis , Región Variable de Inmunoglobulina/biosíntesis , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/metabolismo , Ratones , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/biosíntesis , Distribución Tisular , Trasplante Heterólogo

RESUMEN

The improved tumour to non-tumour ratios needed for effective tumour targeting with antibodies requires that blood background radioactivity is reduced. We investigated the effect of streptavidin as a clearing agent for 125I-labelled biotinylated anti-CEA antibodies in a human colon carcinoma xenograft model. By comparing the biodistribution of the monoclonal antibody A5B7 with four, nine or 22 biotins per antibody molecule, we investigated how the degree of biotinylation of the primary radiolabelled antibody affects its clearance with streptavidin. Limiting the degree of biotinylation limited blood clearance, whereas nine or 22 biotins per antibody molecule resulted in a 13- to 14-fold reduction in blood radioactivity, the streptavidin-biotinylated antibody complexes clearing rapidly via the liver and spleen. Although a reduction in tumour activity was also seen, a 6.6-fold improvement in the tumour to blood ratio was achieved. A comparative study of streptavidin versus second antibody clearance was carried out using the polyclonal antibody PK4S biotinylated with 12 biotins per antibody molecule. This study indicated that second antibody was superior for clearance of the polyclonal antibody, resulting in a larger and faster reduction in blood radioactivity and improved tumour to blood ratios. In this case the primary antibody was polyclonal, and therefore non-uniformity of biotinylation may affect complexation with streptavidin. Therefore, the degree of biotinylation and type of antibody must be carefully considered before the use of streptavidin clearance.

Asunto(s)

Anticuerpos Monoclonales/metabolismo , Neoplasias del Colon/radioterapia , Radioisótopos de Yodo/farmacocinética , Radioinmunoterapia/métodos , Animales , Anticuerpos Monoclonales/sangre , Biotina , Antígeno Carcinoembrionario/inmunología , Neoplasias del Colon/metabolismo , Humanos , Radioisótopos de Yodo/uso terapéutico , Tasa de Depuración Metabólica , Ratones , Ratones Desnudos , Distribución Tisular , Trasplante Heterólogo

RESUMEN

Antibody directed enzyme prodrug therapy (ADEPT) has been studied as a two- and three-phase system in which an antibody to a tumor-associated antigen has been used to deliver an enzyme to tumor sites where it can convert a relatively nontoxic prodrug to a cytotoxic agent. In such a system, it is necessary to allow the enzyme activity to clear from the blood before prodrug injection to avoid toxicity caused by prodrug activation in plasma. To accelerate plasma clearance of enzyme activity, two approaches have been studied. The studies have been performed with a monoclonal anticarcinoembryonic-antigen antibody fragment A5B7-F(ab')2 conjugated to a bacterial enzyme, carboxypeptidase G2 (CPG2), in LS174T xenografted mice. In the first approach, a monoclonal antibody (SB43), directed at CPG2, was used, which inactivates CPG2 in vitro and in vivo. SB43 was galactosylated so that it had sufficient time to form a complex with plasma CPG2, resulting in the inactivation and clearance of the complex from plasma via the carbohydrate-specific receptors in the liver. Injection of SB43gal 19 hours after administration of the radiolabeled conjugate reduced the percentage of injected dose per gram in blood without affecting levels in the tumor. The second approach involved galactosylation of the conjugate so that it cleared rapidly from blood via the asialoglycoprotein receptors in the liver. Localization of the radiolabeled conjugate was achieved by blocking this receptor for about 8 hours with a single injection (8 mg/mouse) of an inhibitor that binds competitively to the receptor. This allowed tumor localization of the conjugate followed by a rapid clearance of the galactosylated conjugate from blood as the inhibitor was consumed. A tumor-to-blood ratio of 45:1 was obtained at 24 hours, which increased to 100:1 at 72 hours after the conjugate injection. These accelerated clearance mechanisms have been applied in antitumor studies in ADEPT.

Asunto(s)

Anticuerpos Monoclonales/farmacología , Anticuerpos Antineoplásicos/farmacología , Antígeno Carcinoembrionario/inmunología , Profármacos/uso terapéutico , Radioinmunoterapia/métodos , gamma-Glutamil Hidrolasa/antagonistas & inhibidores , Animales , Galactosa/metabolismo , Radioisótopos de Yodo/uso terapéutico , Ratones , Ratones Desnudos , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/radioterapia , Factores de Tiempo , gamma-Glutamil Hidrolasa/inmunología

RESUMEN

Insertion of antibody genes into filamentous bacteriophage makes it possible to generate and screen libraries of 10(7) or more antibodies. Each phage expresses an antibody on its surface and contains the corresponding antibody gene. Genes that encode antibodies with desired characteristics are readily selected and their antibodies expressed as soluble proteins in Escherichia coli. We used this system to produce an antibody to carcinoembryonic antigen with higher affinity and better tumour specificity than antibodies currently in use.

Asunto(s)

Anticuerpos Monoclonales/biosíntesis , Anticuerpos Antineoplásicos/biosíntesis , Bacteriófagos/genética , Antígeno Carcinoembrionario/inmunología , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Anticuerpos Antineoplásicos/genética , Anticuerpos Antineoplásicos/inmunología , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Bacteriófagos/inmunología , Biblioteca de Genes , Humanos , Ratones

RESUMEN

Antibody-directed enzyme prodrug therapy (ADEPT) has been studied in a human ovarian carcinoma xenograft grown subcutaneously in nude mice. Radioimmunoassay of supernatants obtained from tumor homogenates showed these to contain carcinoembryonic antigen (CEA). Biodistribution studies with 125I-labeled monoclonal anti-CEA antibody, A5B7, and its F(ab')2 fragment showed localization in these xenografts. The AB57-F(ab')2 fragment conjugated to a bacterial enzyme, carboxypeptidase G2 (CPG2), and, radiolabeled with 125iodine, also localized in the xenografts. The radiolabeled conjugate cleared from blood faster than the antibody alone. The percentage of injected dose per gram in tumor at 24 h postinjection was about fivefold lower than antibody alone. Tumor-to-blood ratio at 72 h after injection of the radiolabeled conjugate was 7 and the tumor-to-normal tissue ratios at this time point ranged from 20 (liver) to 75 (colon). A three-phase ADEPT antitumor study was carried out in which A5B7-F(ab')2-CPG2 was allowed to localize and was followed by accelerated inactivation/clearance of blood CPG2 by a galactosylated anti-CPG2 antibody (SB43gal). A benzoic acid mustard-derived prodrug was injected 24 h after the conjugate, which led to growth delay in this tumor compared to the control untreated group. Further antitumor studies in this model are in progress.

Asunto(s)

Inmunoterapia/métodos , Neoplasias Ováricas/tratamiento farmacológico , Profármacos/uso terapéutico , Animales , Anticuerpos Monoclonales/uso terapéutico , Femenino , Humanos , Inmunoconjugados/uso terapéutico , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Trasplante Heterólogo , gamma-Glutamil Hidrolasa