RESUMO
PURPOSE: To determine the maximum-tolerated dose (MTD) of iodine-131 ((131)I)-labeled 81C6 antitenascin monoclonal antibody (mAb) administered clinically into surgically created resection cavities (SCRCs) in malignant glioma patients and to identify any objective responses with this treatment. PATIENTS AND METHODS: In this phase I trial, newly diagnosed patients with malignant gliomas with no prior external-beam therapy or chemotherapy were treated with a single injection of (131)I-labeled 81C6 through a Rickham reservoir into the resection cavity. The initial dose was 20 mCi and escalation was in 20-mCi increments. Patients were observed for toxicity and response until death or for a minimum of 1 year after treatment. RESULTS: We treated 42 patients with (131)I-labeled 81C6 mAb in administered doses up to 180 mCi. Dose-limiting toxicity was observed at doses greater than 120 mCi and consisted of delayed neurotoxicity. None of the patients developed major hematologic toxicity. Median survival for patients with glioblastoma multiforme and for all patients was 69 and 79 weeks, respectively. CONCLUSION: The MTD for administration of (131)I-labeled 81C6 into the SCRC of newly diagnosed patients with no prior radiation therapy or chemotherapy was 120 mCi. Dose-limiting toxicity was delayed neurologic toxicity. We are encouraged by the survival and toxicity and by the low 2.5% prevalence of debulking surgery for symptomatic radiation necrosis.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Glioma/radioterapia , Imunotoxinas/uso terapêutico , Neoplasias Supratentoriais/radioterapia , Tenascina/imunologia , Adulto , Idoso , Animais , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/imunologia , Terapia Combinada , Feminino , Seguimentos , Glioma/diagnóstico por imagem , Glioma/cirurgia , Humanos , Imunotoxinas/efeitos adversos , Imageamento por Ressonância Magnética , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Neoplasias Supratentoriais/diagnóstico por imagem , Neoplasias Supratentoriais/cirurgia , Análise de Sobrevida , Tomografia Computadorizada de EmissãoRESUMO
Excision and transposition of the Tn5 element in Escherichia coli ordinarily appear to occur by recA-independent mechanisms. However, recA(Prtc) genes, which encode RecA proteins that are constitutively activated to the protease state, greatly enhanced excision and transposition; both events appeared to occur concomitantly and without destruction of the donor DNA. The recombinase function of the RecA protein was not required. Transposition was accompanied by partial, and occasionally full, restoration of the functional integrity of the gene vacated by the excised Tn5. The stimulation of transposition was inhibited by an uncleavable LexA protein and was strongly enhanced by an additional role of the RecA(Prtc) protein besides its mediation of LexA cleavage. To account for the enhanced transposition, we suggest that (i) there may be a LexA binding site within the promoter for the IS50 transposase, (ii) activated RecA may cleave the IS50 transposition inhibitor, and (iii) the transposase may be formed by RecA cleavage of a precursor molecule.
Assuntos
Elementos de DNA Transponíveis , Escherichia coli/genética , Resposta SOS em Genética , Serina Endopeptidases , Proteínas de Bactérias/metabolismo , Bacteriófago lambda/genética , Sequência de Bases , DNA Bacteriano , Genes Bacterianos , Dados de Sequência Molecular , Recombinases Rec A/metabolismo , Proteínas Repressoras/metabolismo , Mapeamento por RestriçãoRESUMO
Vascular leak syndrome (VLS) was originally found to be a major dose-limiting toxicity in humans with cancer treated with several immunotoxins (ITs) containing ricin A chain or blocked ricin. Recently, VLS has also been observed in patients treated with an IT containing the murine monoclonal antibody (MAb) B3 coupled to LysPE38, a recombinant truncated form of Pseudomonas exotoxin (PE) A. Antibody B3 (IgG1k) recognizes LewisY and related carbohydrate epitopes present on many human solid tumors, and B3-LysPE38 showed excellent antitumor activity in nude mice bearing tumors that express the B3 antigen. In the clinical trial, the development of VLS has prevented the administration of the amount of IT necessary to achieve blood levels required for good therapeutic responses. We have now investigated the effects of several PE-based ITs on different human endothelial cell lines to elucidate the mechanism of VLS induced by ITs containing PE. To assess the cytotoxic effect of IT on endothelial cells, various ITs were incubated with cells for 2 or 20 h, and the incorporation of [3H]leucine into protein was measured. The endothelial cells studied were human umbilical vein endothelial cells, human lung-derived microvascular endothelial cells (HUVECs), human adult dermal microvascular endothelial cells, human pulmonary artery endothelial cells, and human aortic endothelial cells. We found that both B3-LysPE38 (LMB-1), a chemical conjugate of MAb B3 with PE38, as well as B3(Fv)-PE38 (LMB-7), a recombinant single chain immunotoxin, inhibited protein synthesis, with 50% inhibitory concentrations between 600 and 1000 ng/ml for 20-h incubation in HUVECs, human lung-derived microvascular endothelial cells, and human adult dermal microvascular endothelial cells but not on human pulmonary artery endothelial cells. The cytotoxic effect was specific since PE38 itself or PE coupled to several other antibodies did not inhibit protein synthesis in these cells even at 10,000 ng/ml. Further evidence that the cytotoxicity of B3-containing ITs is due to specific B3 binding to endothelial cells comes from the fact that the cytotoxicity can be blocked by excess free MAb B3. HUVECs undergo overt morphological changes after treatment with B3-LysPE38 or B3(Fv)PE38. Gaps between the cells are formed after a 20-h exposure but not after 2 h. These studies suggest that VLS in patients is due to capillary damage caused by prolonged exposure to high concentrations of LMB-1.
Assuntos
ADP Ribose Transferases , Anticorpos Monoclonais/efeitos adversos , Toxinas Bacterianas , Síndrome de Vazamento Capilar/etiologia , Endotélio Vascular/efeitos dos fármacos , Exotoxinas/efeitos adversos , Imunotoxinas/efeitos adversos , Antígenos do Grupo Sanguíneo de Lewis/imunologia , Fatores de Virulência , Especificidade de Anticorpos , Células Cultivadas/efeitos dos fármacos , Exotoxinas/química , Humanos , Imunotoxinas/química , Proteínas/efeitos dos fármacos , Proteínas/metabolismo , Exotoxina A de Pseudomonas aeruginosaRESUMO
A single-chain antibody fragment, MR1(scFv), with specific binding to epidermal growth factor receptor-vIII (EGFRvIII), was produced, radiolabeled, and evaluated for biodistribution in human glioma-bearing athymic mice. The mutant receptor EGFRvIII has a deletion in its extracellular domain that results in the formation of a new, tumor-specific antigen found in glioblastomas, breast carcinomas, and other tumors. The scFv molecule, designed as V(H)-(Gly4-Ser)3-V(L), was expressed in Escherichia coli in inclusion body form; recovered scFv fragments were properly refolded in redox-shuffling buffer. Size-exclusion chromatography of purified scFv demonstrated a protein monomer of Mr 26,000. Labeling was performed using N-succinimidyl 5-[125I]iodo-3-pyridinecarboxylate (SIPC) or Iodogen to specific activities of 0.5-2.0 mCi/mg, with yields of 35-50% and 45-70%, respectively. The immunoreactive fraction (IRF) of the labeled MR1(scFv) was 65-80% when SIPC was used and 50-55% when Iodogen was used. The affinity (K(A)) of MRI(scFv) for EGFRvIII was 4.3 x 10(7) +/- 0.1 x 10(7) M(-1) by BIAcore analysis, and it was 1.0 x 10(8) +/- 0.1 x 10(8) M(-1) and by Scatchard analysis versus EGFRvIII-expressing cells. After incubation at 37 degrees C for 24 h, the binding affinity was maintained, and the IRF was maintained at 60-70%. The specificity of MR1(scFv) for EGFRvIII was demonstrated in vitro by incubation of radiolabeled MR1(scFv) with the EGFRvIII-expressing U87MG.deltaEGFR cell line in the presence or absence of competing unlabeled MR1(scFv) or anti-EGFRvIII MAbs L8A4 and H10. In biodistribution studies using athymic mice bearing s.c. U87MG.deltaEGFR tumor xenografts, animals received intratumoral or i.v. infusions of paired-label [125I]SIPC-MR1(scFv) and [131I]SIPC-anti-Tac(scFv) as a control. When given by the intratumoral route, MR1(scFv) retained high tumor uptakes of 85% injected dose per gram of tissue at 1 h and 16% injected dose per gram of tissue at 24 h following administration. Specific: control scFv tumor uptake ratios of more than 20:1 at 24 h demonstrated specific localization of MR1(scFv). The excellent tumor retention of MR1(scFv), combined with its rapid clearance from normal tissues, resulted in high tumor:normal organ ratios.
Assuntos
Especificidade de Anticorpos , Receptores ErbB/imunologia , Glioma/terapia , Região Variável de Imunoglobulina/farmacologia , Sequência de Aminoácidos , Animais , Ligação Competitiva , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Receptores ErbB/metabolismo , Feminino , Glioma/imunologia , Glioma/metabolismo , Humanos , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/metabolismo , Radioisótopos do Iodo , Camundongos , Dados de Sequência Molecular , Transplante de Neoplasias , Dobramento de Proteína , Distribuição TecidualRESUMO
Growth factor receptors provide unique opportunities for development of targeted anticancer therapy. Members of the type I receptor tyrosine kinase family, including epidermal growth factor (EGF) receptor (EGFR) and ErbB-2/neu, are often overexpressed in various human cancer cells, including breast. Recently, it has been shown that both ErbB-3 and ErbB-4 are receptors for heregulin (HRG)/Neu differentiation factor. Eight chimeric toxins composed of the extracellular and EGF-like domains of four different HRG isoforms and truncated Pseudomonas exotoxin (PE38KDEL) were constructed. The fusion proteins exhibited activity similar to the native HRG in inducing ErbB receptors phosphorylation. The EGF-like domain of HRG13 and HRGbeta2 fused to PE38KDEL showed the highest cytotoxic activity, with a IC50 of < or = 0.001 ng/ml. The alpha isoforms that were fused to PE38KDEL were 100-fold less active than the beta isoforms. The HRG-Pseudomonas exotoxin (PE) toxins show extremely high activity against cells expressing ErbB-4 receptor, alone or together with other members of the ErbB receptor family. Cells that do not express ErbB-4 but express ErbB-3 receptor, together with the ErbB-2 or EGFR, exhibited moderate sensitivity to HRG-PE toxins. HRG-PE toxins have little or no activity against cells expressing EGFR, ErbB-2, or ErbB-3 alone. More than an 80% tumor regression was achieved by intratumor injection of 1 microg of fusion proteins per day for 5 days. Continuous i.p. administration of EGF-like domain of HRGbeta1-PE38KDEL for 7 days via a miniosmotic pump at a dose of 40 microg/kg/day inhibited the growth of ErbB-4 receptor positive but not ErbB-4 receptor negative cell lines in athymic nude mice. We conclude that there is therapeutic potential of HRG-PE toxins in the therapy of cancers overexpressing the ErbB-4 or ErbB-2 plus ErbB-3 receptors.
Assuntos
Antineoplásicos/farmacologia , Receptores ErbB/efeitos dos fármacos , Exotoxinas/farmacologia , Genes erbB/efeitos dos fármacos , Glicoproteínas/farmacologia , Proteínas Proto-Oncogênicas/efeitos dos fármacos , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Carcinoma/tratamento farmacológico , Ensaio de Unidades Formadoras de Colônias , Exotoxinas/farmacocinética , Exotoxinas/uso terapêutico , Feminino , Glicoproteínas/farmacocinética , Glicoproteínas/uso terapêutico , Humanos , Imunotoxinas/farmacocinética , Imunotoxinas/farmacologia , Imunotoxinas/uso terapêutico , Camundongos , Camundongos Nus , Fosforilação , Receptor ErbB-3 , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes de Fusão/uso terapêutico , Neoplasias Gástricas/tratamento farmacológico , Células Tumorais Cultivadas/efeitos dos fármacos , Tirosina/metabolismoRESUMO
The incidence of neoplastic meningitis is on the rise. Neoplastic meningitis can result from a direct seeding of the neuraxis by primary brain tumors or by hematogeneous spread of systemic solid tumors. A frequent genetic alteration in primary brain tumors such as gliomas is an in-frame deletion in the epidermal growth factor receptor (EGFR) gene EGFRvIII, which brings together what were normally distant polypeptide sequences in the intact receptor. A novel glycine is formed at the fusion junction, resulting in a unique and tumor-specific target. By using phage display, we have isolated a single-chain antibody specific for the EGFRvIII mutation and expressed it with a modified form of the Pseudomonas exotoxin to form the immunotoxin MR1scFvPE38KDEL (MR-1). The multiple dose toxicity and therapeutic efficacy of MR-1 immunotoxin were tested in an athymic rat model of neoplastic meningitis. The maximally tolerated doses in non-tumor-bearing rats were three doses of 3 microg each. For therapeutic studies, the target was a neoplastic meningitis induced by intrathecal inoculation of the EGFRvIII-expressing human glioma U87MG.deltaEGFR. A dose escalation study compared the survival of three equal doses of 1, 2, and 3 microg of MR-1 immunotoxin with saline or 3 microg of the control immunotoxin specific for the interleukin 2 receptor, anti-Tac. All animals treated with three doses of saline or 3 microg of anti-Tac died, with median survival of 7 and 10 days, respectively. There were 75% (six of eight) long-term survivors in the group treated with three doses of 1 microg and 57% (four of seven) long-term survivors in the groups treated with three doses of either 2 or 3 microg of MR-1 immunotoxin. None of the MR-1 immunotoxin-treated groups reached median survival by the termination of the study at 53 days. Therefore, median survival was estimated to be >53 days, resulting in an estimated increase in median survival of >657% compared with saline and 430% versus anti-Tac. Compartmental therapy with three doses of 2 microg of MR-1 immunotoxin is effective in the treatment of EGFRvIII-expressing neoplastic meningitis. This dose was found to have no clinical or histopathological effects on non-tumor-bearing animals. MR-1 immunotoxin is, therefore, considered specific and safe within its therapeutic window. Phase I clinical trials for tumors invading the intrathecal space that express the EGFRvIII target should be initiated.
Assuntos
ADP Ribose Transferases , Toxinas Bacterianas , Receptores ErbB/imunologia , Receptores ErbB/metabolismo , Exotoxinas/farmacologia , Região Variável de Imunoglobulina/farmacologia , Imunotoxinas/farmacologia , Neoplasias Meníngeas/tratamento farmacológico , Fatores de Virulência , Animais , Especificidade de Anticorpos , Receptores ErbB/biossíntese , Receptores ErbB/genética , Exotoxinas/toxicidade , Feminino , Humanos , Região Variável de Imunoglobulina/toxicidade , Imunotoxinas/toxicidade , Injeções Espinhais , Neoplasias Meníngeas/metabolismo , Camundongos , Camundongos Nus , Mutação , Inibidores da Síntese de Proteínas/farmacologia , Ratos , Ratos Nus , Células Tumorais Cultivadas , Exotoxina A de Pseudomonas aeruginosaRESUMO
Cell surface receptors are attractive candidates for targeted therapy of cancer. Growth factors and their receptors play important roles in the regulation of cell division, development, and differentiation. Among those, the epidermal growth factor receptor (EGFR) was the first identified to be amplified and/or rearranged in malignant gliomas. The most common rearranged form, EGFR type III variant (EGFRvIII), has a deletion in its extracellular domain that results in the formation of a new, tumor-specific target found in glioblastoma multiforme, as well as in breast, ovarian, prostate, and lung carcinomas. Monoclonal antibodies have been developed with specific activity against this mutant receptor. These antibodies are internalized into the cell after receptor binding. Specific antibodies, either unarmed or armed with cytotoxic agents, including radioisotopes and toxins, have shown a promising role for EGFRvIII as a target for brain tumor therapy.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Anticorpos Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/terapia , Receptores ErbB/imunologia , Glioblastoma/imunologia , Glioblastoma/terapia , Imunoterapia Ativa/métodos , Animais , Anticorpos Monoclonais/toxicidade , Anticorpos Antineoplásicos/toxicidade , Antineoplásicos/imunologia , Antineoplásicos/toxicidade , Humanos , Imunotoxinas/uso terapêutico , Imunotoxinas/toxicidadeRESUMO
Epidermal growth factor receptor (EGFR) is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC) where aberrant signaling downstream of this receptor contributes to tumor growth. EGFR variant III (EGFRvIII) is the most commonly altered form of EGFR and contains a truncated ligand-binding domain. We previously reported that EGFRvIII is expressed in up to 40% of HNSCC tumors where it is associated with increased proliferation, tumor growth and chemoresistance to antitumor drugs including the EGFR-targeting monoclonal antibody cetuximab. Cetuximab was FDA-approved in 2006 for HNSCC but has not been shown to prevent invasion or metastasis. This study was undertaken to evaluate the mechanisms of EGFRvIII-mediated cell motility and invasion in HNSCC. We found that EGFRvIII induced HNSCC cell migration and invasion in conjunction with increased signal transducer and activator of transcription 3 (STAT3) activation, which was not abrogated by cetuximab treatment. Further investigation showed that EGF-induced expression of the STAT3 target gene HIF1-α, was abolished by cetuximab in HNSCC cells expressing wild-type EGFR under hypoxic conditions, but not in EGFRvIII-expressing HNSCC cells. These results suggest that EGFRvIII mediates HNSCC cell migration and invasion by increased STAT3 activation and induction of HIF1-α, which contribute to cetuximab resistance in EGFRvIII-expressing HNSCC tumors.
Assuntos
Carcinoma de Células Escamosas/patologia , Receptores ErbB/fisiologia , Neoplasias de Cabeça e Pescoço/patologia , Invasividade Neoplásica , Fator de Transcrição STAT3/metabolismo , Carcinoma de Células Escamosas/genética , Receptores ErbB/genética , Neoplasias de Cabeça e Pescoço/genética , HumanosAssuntos
Receptores ErbB/imunologia , Imunoterapia/métodos , Neoplasias/imunologia , Neoplasias/terapia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Receptores ErbB/genética , Regulação Neoplásica da Expressão Gênica , Glioblastoma/imunologia , Glioblastoma/terapia , Humanos , Imunotoxinas/imunologia , Imunotoxinas/uso terapêutico , MutaçãoRESUMO
The LexA protein of Escherichia coli represses expression of a variety of genes that, by definition, constitute the SOS regulon. Genetic evidence suggests that Tn5 transposition is also regulated by the product of the lexA gene (C.-T. Kuan, S.-K. Liu, and I. Tessman, Genetics 128:45-57, 1991). We now show that the LexA protein represses expression of the tnp gene, located in the IS50R component of Tn5, which encodes a transposase, and that LexA does not repress expression of the IS50R inh gene, which encodes an inhibitor of transposition. Elimination of LexA resulted in increased expression of the tnp gene by a factor of 2.7 +/- 0.4, as indicated by the activity of a lacZ gene fused to the tnp gene. LexA protein retarded the electrophoretic movement of a 101-bp segment of IS50R DNA that contained a putative LexA protein-binding site in the tnp promoter; the interaction between the LexA repressor and the promoter region of the tnp gene appears to be relatively weak. These features show that the IS50R tnp gene is a member of the SOS regulon.
Assuntos
Proteínas de Bactérias/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Nucleotidiltransferases/genética , Serina Endopeptidases , Proteínas de Bactérias/metabolismo , Sequência de Bases , Elementos de DNA Transponíveis/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/fisiologia , Proteínas Recombinantes de Fusão/biossíntese , Resposta SOS em Genética/genética , Transposases , beta-Galactosidase/genéticaRESUMO
We have shown that excision and transposition of Tn5 in Escherichia coli are greatly increased by recA(Prtc) genes, which encode constitutively activated RecA proteins (C.-T. Kuan, S.-K. Liu, and I. Tessman, Genetics 128:45-57, 1991). Contrary results, showing a significant decrease in Tn5 transposition under SOS conditions, were subsequently reported (M. D. Weinreich, J. C. Makris, and W. S. Reznikoff, J. Bacteriol. 173:6910-6918, 1991). We have extended our studies to examine the following: (i) transposition of Tn5 from sites in the phoA, phoB, proC, trpD, and ilvD genes; (ii) the effect of gene transcription; (iii) the comparative effect of dinD+ and dinD(Def) alleles; (iv) the use of a mating-out assay of transposition; (v) the effect of a recA(Prtc) allele located at the normal chromosomal site; and (vi) the effect at 41.5 degrees C of the recA441(Prtc) allele. The new results fully confirm our previous conclusions, including the fact that the frequency of Tn5 transposition under constitutive SOS conditions is site dependent.
Assuntos
Elementos de DNA Transponíveis/genética , Escherichia coli/genética , Genes Bacterianos/genética , Recombinases Rec A/genética , Transcrição Gênica , Alelos , Cruzamentos Genéticos , Fator F/genéticaRESUMO
PE35/e23(dsFv)KDEL is a recombinant immunotoxin composed of a recombinant form of Pseudomonas exotoxin that does not need proteolytic activation and a disulfide-stabilized Fv fragment of the anti-erbB2 monoclonal antibody e23. In this molecule, the variable heavy (V H) domain is inserted near the carboxyl terminus of PE at position 607 and the variable light (V L) domain is connected to the V H domain by a disulfide bond engineered into the framework region. The disulfide bond forms between cysteines introduced at position 44 of V H and position 99 of V L [Reiter et al. (1994) J. Biol. Chem. 269, 18327-18331]. In contrast to other PE-derived Fv fusion proteins, this type of recombinant toxin does not need proteolytic activation of the toxin domain. PE35/e23(dsFv)KDEL is very cytotoxic toward erbB2 antigen-expressing N87 cells (IC50 = 0.8 ng/mL) despite the fact that it binds to the erbB2 protein only 25% as well as e23(dsFv)PE38KDEL, in which the dsFv moiety is located at the amino terminus of the toxin. The lower binding affinity is probably due to interference by domain III of PE with the amino terminus of e23(V H), possibly where the antigen binding sites are located. Nevertheless, the specificity of immunotoxin is still retained, and it is very stable at 37 degrees C. Because of its small size, stability, and activity without proteolytic processing, this immunotoxin may be advantageous for tumor treatment. PE35/e23(dsFv)KDEL was also used to gain information about whether reduction of the disulfide bonds connecting V H and V L occur in the endoplasmic reticulum (ER) or in a proximal compartment. To do this, we switched the ER retention sequence KDEL from the toxin--V H subunit to the V L subunit. Our results suggest that reduction of the disulfide bond connecting the dsFv heterodimer occurs before the immunotoxin reaches the ER, where translocation to the cytosol appears to occur.
Assuntos
ADP Ribose Transferases , Endopeptidases/metabolismo , Exotoxinas/química , Imunotoxinas/química , Fatores de Virulência , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Sequência de Bases , Ligação Competitiva , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Primers do DNA , Dissulfetos/química , Dissulfetos/metabolismo , Exotoxinas/metabolismo , Exotoxinas/toxicidade , Humanos , Região Variável de Imunoglobulina , Imunotoxinas/metabolismo , Imunotoxinas/toxicidade , Cinética , Dados de Sequência Molecular , Mutagênese Insercional , Plasmídeos , Reação em Cadeia da Polimerase , Inibidores da Síntese de Proteínas/toxicidade , Receptor ErbB-2/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Exotoxina A de Pseudomonas aeruginosaRESUMO
B1(dsFv)-PE33 is a recombinant immunotoxin composed of a mutant form of Pseudomonas exotoxin (PE) that does not need proteolytic activation and a disulfide-stabilized Fv fragment of the anti-Lewis(y) monoclonal antibody B1, which recognizes a carbohydrate epitope on human carcinoma cells. In this molecule, amino acids 1-279 of PE are deleted and domain Ib (amino acids 365-394) is replaced by the heavy chain variable region (VH) domain of monoclonal antibody B1. The light chain (VL) domain is connected to the VH domain by a disulfide bond. This recombinant toxin, termed B1(dsFv)-PE33, does not require proteolytic activation and it is smaller than other immunotoxins directed at Lewis(y), all of which require proteolytic activation. Furthermore, it is more cytotoxic to antigen-positive cell lines. B1(dsFv)-PE38 has the highest antitumor activity of anti-Lewis(y) immunotoxins previously constructed. B1(dsFv)-PE33 caused complete regression of tumors when given at 12 micrograms/kg (200 pmol/kg) every other day for three doses, whereas B1(dsFv)-PE38 did not cause regressions at 13 micrograms/kg (200 pmol/kg). By bypassing the need for proteolytic activation and decreasing molecular size we have enlarged the therapeutic window for the treatment of human cancers growing in mice, so that complete remissions are observed at 2.5% of the LD50.
Assuntos
ADP Ribose Transferases , Adenocarcinoma/tratamento farmacológico , Antineoplásicos/toxicidade , Antineoplásicos/uso terapêutico , Toxinas Bacterianas , Exotoxinas/toxicidade , Exotoxinas/uso terapêutico , Imunotoxinas/toxicidade , Imunotoxinas/uso terapêutico , Fatores de Virulência , Animais , Anticorpos Monoclonais , Sequência de Bases , Neoplasias da Mama , Sobrevivência Celular/efeitos dos fármacos , Primers do DNA , Exotoxinas/biossíntese , Feminino , Humanos , Células L , Leucemia de Células T , Antígenos do Grupo Sanguíneo de Lewis/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase , Neoplasias da Próstata , Pseudomonas aeruginosa , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/uso terapêutico , Proteínas Recombinantes/toxicidade , Neoplasias Gástricas , Transplante Heterólogo , Células Tumorais Cultivadas , Exotoxina A de Pseudomonas aeruginosaRESUMO
Pseudomonas exotoxin A (PE) is a three-domain protein in which domain Ia is involved in recognition of receptors on eukaryotic target cells, domain II promotes translocation of PE into the cytosol, and domain III enzymatically ADP-ribosylates elongation factor 2. Modification of proteins with polyethylene glycol (PEG) has been shown to prolong circulating plasma lifetime and may reduce or eliminate immunogenicity. However, in the case of toxins, PEG may interfere with or block toxin activity. To investigate the effect of polyethylene glycolation on specific residues located on the surface of PE domain II, we substituted cysteine, for each of the five most exposed surface amino acids (H276, E282, N306, R313, and E327) in domain II. These cysteines can serve as unique sites for PEG modification. The PE-Cys proteins retained most of their cytotoxicity even when the free sulfhydryl group was blocked by 5,5'-dithiobis(nitrobenzoic acid) or glutathione. When the PE-Cys proteins were conjugated with ovalbumin using a cleavable disulfide linkage, cytotoxicity was retained, but it was lost with a non-cleavable thioether linkage. In contrast, cytotoxicity was maintained when PE-Cys mutants were coupled to 5- or 20-kDa mPEG, using either a disulfide or a thioether linkage. Unexpectedly in some cases, the thioether conjugate was more active than the disulfide linkage. Pharmacokinetic studies on one of the polyethylene-glycolated molecules (R313C) showed that the mean residence time (t 1/2) was prolonged to 72 min, compared to 20 min for unpolyethylene glycolated PE-Cys(R313C). These studies show it is possible to derivatize PE at specific residues in domain II, maintain significant cytotoxic activity, and alter pharmacokinetics. These studies also suggest that large mPEG molecules can be translocated to the cytosol while still attached to domain II of PE.
Assuntos
ADP Ribose Transferases , Toxinas Bacterianas/genética , Cisteína/metabolismo , Exotoxinas/genética , Mutação , Polietilenoglicóis/farmacologia , Fatores de Virulência , Animais , Toxinas Bacterianas/química , Toxinas Bacterianas/farmacocinética , Toxinas Bacterianas/toxicidade , Células Cultivadas , Cisteína/efeitos dos fármacos , Cisteína/genética , Exotoxinas/química , Exotoxinas/farmacocinética , Exotoxinas/toxicidade , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Dobramento de Proteína , Pseudomonas aeruginosa/metabolismo , Células Tumorais Cultivadas , Exotoxina A de Pseudomonas aeruginosaRESUMO
EGFRvIII is the most common deletion variant of the epidermal growth factor receptor and is found in cancers of the brain, breast, ovary, and lung. The complete absence of the receptor in healthy tissues makes it an ideal tumor marker. We sought to design a peptide ligand against EGFRvIII for development as a diagnostic imaging agent. We used the concept of hydropathic complementarity to search for sequences whose amino acid sidechains display a reciprocal pattern of hydropathicity to those of the deletion junction of EGFRvIII. The resulting peptide (PEPHC1) was synthesized and tested for binding to EGFRvIII and EGFR. In in vitro assays, PEPHC1 bound the recombinant EGFRvIII extracellular domain or full-length EGFRvIII solubilized from cell membranes in preference to native EGFR. These results demonstrate the utility of hydropathic complementarity as a basis for the design of highly specific ligands that may prove useful as tumor-targeting agents.
Assuntos
Receptores ErbB/genética , Peptídeos/química , Células 3T3 , Sequência de Aminoácidos , Animais , DNA Complementar , Camundongos , Dados de Sequência Molecular , Peptídeos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
Combinatorial variation of CDR3 of V(H) and V(L), followed by phage display, was used to select affinity mutants of the parental anti-epidermal growth factor receptor-vIII (EGFRvIII) scFv MR1. One mutant, MR1-1(scFv), had increased specific binding affinity for EGFRvIII. It was produced and radiolabeled, and its biodistribution was evaluated in human glioma-bearing athymic mice. MR1-1 targeted the same EGFRvIII epitope as MR1 with an approximately 15-fold higher affinity (K(d) = 1.5 x 10(-9) M) measured by surface resonance analysis. Labeling with (131)I or (125)I was performed, and the immunoreactive fraction of the labeled MR1-1(scFv) was 50% to 55%. After incubation at 37 degrees C for 4 days, the binding affinity was maintained at 60% of initial levels. The specificity of MR1-1 for EGFRvIII was demonstrated in vitro by flow cytometry and incubation of FITC-labeled scFv with the EGFRvIII-expressing U87MG. DeltaEGFR cell line or with the EGFRvIII-negative U87MG cell line in the presence or absence of competing unlabeled MR1-1(scFv). We also investigated the internalization and processing of MR1-1 compared with MR1; MR1-1 exhibited levels of both cell surface retention and internalization up to 5 times higher than those by MR1. In biodistribution studies performed in athymic mice bearing s.c. U87MG. DeltaEGFR tumor xenografts, animals received paired-label intratumoral infusions of (131)I-labeled MR1-1(scFv) and (125)I-labeled MR1(scFv). Our results showed an up to 244% +/- 77% increase in tumor uptake for MR1-1 compared with that for MR1. The improved tumor retention of MR1-1(scFv) combined with its rapid clearance from normal tissues also resulted in sustained higher tumor:normal organ ratios. These results suggest that the improved affinity of MR1-1 can significantly impact in vivo glioma-specific targeting and immunotherapy.