RESUMO
Human immunoglobulin transgenic mice provide a method of obtaining human monoclonal antibodies (Mabs) using conventional hybridoma technology. We describe a novel strain of human immunoglobulin transgenic mice and the use of this strain to generate multiple high-avidity human sequence IgG kappa Mabs directed against a human antigen. The light chain transgene is derived in part from a yeast artificial chromosome clone that includes nearly half of the germline human V kappa region. In addition, the heavy-chain transgene encodes both human mu and human gamma 1 constant regions, the latter of which is expressed via intratransgene class switching. We have used these animals to isolate human IgG kappa Mabs that are specific for the human T-cell marker CD4, have high binding avidities, and are immunosuppressive in vitro. The human Mab-secreting hybridomas display properties similar to those of wild-type mice including stability, growth, and secretion levels. Mabs with four distinct specificities were derived from a single transgenic mouse, consistent with an extensive diversity in the primary repertoire encoded by the transgenes.
Assuntos
Anticorpos Monoclonais/biossíntese , Afinidade de Anticorpos , Imunoglobulina G/imunologia , Cadeias kappa de Imunoglobulina/imunologia , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Antígenos CD4/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Hibridomas , Teste de Cultura Mista de Linfócitos , Camundongos , Camundongos Transgênicos , Linfócitos T/imunologiaRESUMO
In vivo efficacy testing of monoclonal antibody-based drugs specific for human leukemias is hampered by the paucity of suitable animal models, due in part to the inability of many anti-human monoclonal antibodies to cross-react with antigens expressed in animal tissues or cells. Moreover, human leukemic cells have proven difficult to establish in immunosuppressed mice except as solid tumors. We report here the establishment of a murine model for human leukemia displaying features of human disease, such as growth of malignant cells and localization of such cells to lymphoid compartments, and the effective depletion of leukemic cells from these mice by an immunoconjugate. Human T-leukemia cells (CEM) injected into cyclophosphamide-pretreated NIH-III mice engrafted in all mice (n = 41), with CEM cells detected in the bone marrow, spleen, and blood 4 weeks after injection. There was no evidence of solid tumors. Treatment of CEM-engrafted mice with 4A2-RTA30, an immunoconjugate of an anti-CD7 monoclonal antibody and ricin A chain (RTA30), resulted in a 100- to 200-fold overall depletion of CEM cells from the spleen and the bone marrow (P less than 0.02). This depletion was specific and toxin-dependent, as a control immunoconjugate had no demonstrable effect (P greater than 0.5). Depletion of CEM cells was also observed after treatment with unconjugated anti-CD7 mAb, but this effect was not significantly different from controls (P greater than 0.1). Therefore, significant depletion of CEM cells required the presence of the ricin A chain moiety. Further investigations revealed that CEM cells recovered from NIH-III mice expressed less CD7 antigen, but remained sensitive to subsequent in vitro exposure to 4A2-RTA30. In conclusion, we have established a model for studying the efficacy of immunoconjugates and have successfully depleted human T-leukemic cells from lymphoid tissues in immunodeficient mice by treatment with an anti-CD7-RTA30 immunoconjugate.
Assuntos
Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Imunotoxinas/uso terapêutico , Leucemia-Linfoma de Células T do Adulto/terapia , Ricina/uso terapêutico , Animais , Anticorpos Monoclonais/uso terapêutico , Antígenos CD/análise , Antígenos CD7 , Linhagem Celular , Ciclofosfamida/farmacologia , Esquema de Medicação , Avaliação Pré-Clínica de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Antígenos de Histocompatibilidade/análise , Humanos , Terapia de Imunossupressão , Imunotoxinas/toxicidade , Antígenos Comuns de Leucócito , Masculino , Glicoproteínas de Membrana/análise , Camundongos , Camundongos Endogâmicos , Transplante de Neoplasias/métodos , Ricina/toxicidade , Transplante HeterólogoRESUMO
The sigma subunit of RNA polymerase is responsible for specific initiation of RNA synthesis at promoter sites on DNA. sigma dissociates shortly after initiation. Photoaffinity-labeling experiments performed on transcription complexes with two different DNA promoters, which have highly homologous control sequences upstream from the transcribed regions, have revealed that the sigma subunit of RNA polymerase is contacted by the 5' ends of quite different lengths of nascent RNA in each transcription complex. On the other hand, the labeling of subunits beta beta' is quite similar for both promoters, and the alpha subunit is not labeled in either case. The results of transcription experiments on the phage lambda PR promoter show that sigma can be photoaffinity labeled by RNA chains that are 9-13 nucleotides long and thus remains associated with the core enzyme at least to that point. But on the A1 promoter of phage T7 DNA, photoaffinity labeling of sigma ceases with the trinucleotide. Thus release of sigma from the vicinity of nascent RNA depends not merely on the length but on the sequence of the transcript. For the T7 A1 promoter, sigma labeling ceases while the leading end of the RNA is still base paired to the DNA template; thus, it appears that there is at least one site on the enzyme that interacts with the growing transcript/template hybrid, in a sequence-dependent way, to effect sigma release.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Bacteriófago lambda/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Regiões Promotoras Genéticas , Fagos T/genética , Transcrição Gênica , Escherichia coli/enzimologia , Escherichia coli/genética , Substâncias Macromoleculares , Especificidade da EspécieRESUMO
A photoaffinity-protection technique has been developed to study the accessibility of the leading (5') end of nascent RNA as it passes through the transcription complex formed by Escherichia coli RNA polymerase and phage T7 DNA. The macromolecules contacted by the leading (5') end of the growing RNA chain in the transcription complex have been determined previously by photoaffinity labeling experiments using aryl azides attached to the leading end of nascent RNA [Hanna, M. M., & Meares, C. F. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4238-4242]. By using thiols to reduce accessible photoprobes, we have modified the photoaffinity technique so that it tests the accessibility of the leading end of nascent RNA to small molecules in solution, as a function of RNA chain length. We examined in detail RNA molecules containing 11-50 nucleotides, whose 5' ends label the beta and beta' enzyme subunits with good yield. The thiol's accessibility to the leading end of each transcript was determined by comparing the RNAs cross-linked to beta beta' in thiol-treated samples to controls not treated with thiol. Incubation with 1 mM dithiothreitol for 5 min reduced approximately 36% of the 5'-azides on RNAs 11-13 bases long and approximately 43% on RNAs 28-37 bases long but practically none of the 5'-azides on RNAs 40-43 bases long. Also notable was the reduction of 34 +/- 1% of the 5'-azides on RNA 12 bases long but only 14 +/- 2% on the 14-base RNA; on the T7 A1 promoter, the leading end of the transcript diverges from the DNA template when the chain is between 12 and 14 bases long.
Assuntos
Escherichia coli/genética , RNA Bacteriano/genética , RNA Viral/genética , Fagos T/genética , Transcrição Gênica , DNA Viral/genética , RNA Polimerases Dirigidas por DNA/genética , Substâncias MacromolecularesRESUMO
Ribosomal inactivating proteins such as gelonin (Gel) and ricin A chain (RTA) conjugated to MoAbs bind to specific target cells, and upon internalization inhibit protein synthesis, ultimately resulting in cell death. We report here that Gel anti-T cell MoAb conjugates are more cytotoxic than RTA conjugates when tested against human peripheral blood mononuclear cells (PBMC). This increased cytotoxicity is observed whether Gel is conjugated to the anti-T cell MoAb or to an anti-mouse immunoglobulin Fab' fragment which then binds to the murine anti-human T cell MoAb. Gel conjugates are not only effective at lower concentrations, but also produce a greater extent of inhibition of cellular proliferation. Moreover, a 10 min exposure to a Gel conjugate is as effective as a 90 h exposure to an RTA conjugate. When part of anti-T cell F(ab')2 or Fab' conjugates, Gel affects the early steps in cellular intoxication more than RTA; Gel conjugates bind more avidly and accelerate the modulation of antigen. In contrast, when part of whole IgG conjugates, Gel does not affect the binding to or modulation of surface antigen compared with RTA, while it does increase conjugate cytotoxicity. These observations suggest that Gel may be delivered more efficiently into the cytosol than RTA. A divergent intracellular pathway for Gel is also supported by the inability of chemical potentiators, which strongly enhance RTA potency, to affect Gel potency. These properties of Gel might also be advantageous for immunoconjugates made with other MoAbs or receptor-binding molecules.
Assuntos
Citotoxicidade Imunológica , Imunotoxinas/farmacologia , Proteínas de Plantas/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Soro Antilinfocitário/farmacologia , Ligação Competitiva , Humanos , Imunoterapia , Técnicas In Vitro , Camundongos , Proteínas Inativadoras de Ribossomos Tipo 1 , Ricina/farmacologiaRESUMO
A synthetic gene for the Aspergillus protein toxin mitogillin has been synthesized and expressed in Escherichia coli. The recombinant mitogillin is a potent inhibitor of protein synthesis in vitro with an IC50 of 9.7 pM. Immunoconjugates of recombinant mitogillin derivatized with S-acetylmercaptosuccinic anhydride and 5-methyl-2-iminothiolane modified H65 antibody kill T cell lines and peripheral blood mononuclear cells expressing the human CD5 surface antigen. Native mitogillin contains 4 cysteine residues which form two disulfide pairs (Fernandez-Luna, J. L., Lopez-Otin, C., Soriano, F., and Mendez, E. (1985) Biochemistry 24, 861-867). Three derivatives of mitogillin have been assembled which substitute alanine residues for cysteine residues 5, 147, or 5 and 147. Each of these molecules retains the ability to inhibit protein synthesis in vitro with at most a 2-fold reduction in activity. The derivative mitogillinC147A can be conjugated to 5-methyl-2-iminothiolane- modified H65 antibody directly without pretreatment with S-acetylmercaptosuccinic anhydride, and the immunoconjugate is active against HSB2 cells. Genetic manipulation of toxin genes to expose an accessible cysteine residue into a recombinant product can thus be used to generate immunotoxins without initial derivatization by nonspecific cross-linking reagents.
Assuntos
Alérgenos , Antibióticos Antineoplásicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Proteínas Fúngicas/farmacologia , Genes Sintéticos , Imunotoxinas/toxicidade , Ribonucleases , Linfócitos T/efeitos dos fármacos , Adulto , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Antígenos de Plantas , Aspergillus/genética , Sequência de Bases , Linhagem Celular , Células Cultivadas , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Globinas/genética , Humanos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Plasmídeos , Coelhos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Mapeamento por Restrição , Reticulócitos/metabolismo , Linfócitos T/citologiaRESUMO
A human peripheral blood lymphocyte-reconstituted severe combined immunodeficient (hu-PBL-SCID) mouse model was used to compare in vivo efficacy of immunoconjugates directed against the CD5 antigen present on human T-cells. Four anti-CD5 immunoconjugates were tested, composed of chimeric human-mouse (cH65) F(ab')2 or Fab' fragments chemically linked to recombinant gelonin (rGEL) or the 30,000 M(r) glycoform of ricin A chain (RTA30). Immunoconjugate treatment was initiated approximately 3 weeks after PBL transplantation and consisted of five consecutive daily bolus i.v. injections. Efficacy was subsequently assessed by quantitation of human T-cells in spleens, blood and peritoneal lavage fluid using 3-color flow cytometry. cH65 F(ab')2- and cH65 Fab'-rGEL conjugates were essentially equally effective at depleting human T-cells from SCID mouse tissues, suggesting that bivalent binding is not required for efficacy when rGEL is the cytotoxic moiety. Treatment with unconjugated F(ab')2, unconjugated Fab' or a Fab-rGEL immunoconjugate of irrelevant binding specificity did not result in a significant depletion of T-cells, demonstrating that the cytotoxic moiety and a relevant human T-cell binding moiety are both required for efficacy. In contrast to the results observed with the rGEL conjugates, cH65 Fab'-RTA30 was not as effective as cH65 F(ab')2-RTA30 in depleting human T-cells from SCID mouse tissues. This paralleled in vitro findings in a human PBMC cytotoxicity assay, which demonstrated that cH65 Fab'-RTA30 was 17-fold less potent than cH65 F(ab')2-RTA30 and approximately 50-fold less potent than the rGEL conjugates.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Imunotoxinas/farmacologia , Animais , Antígenos CD/imunologia , Antígenos CD5 , Citometria de Fluxo , Humanos , Fragmentos Fab das Imunoglobulinas , Transfusão de Leucócitos , Camundongos , Camundongos SCID , Cavidade Peritoneal/citologia , Fito-Hemaglutininas/farmacologia , Proteínas de Plantas , Receptores de Interleucina-2/efeitos dos fármacos , Proteínas Inativadoras de Ribossomos Tipo 1 , Ricina , Baço/citologia , Linfócitos T/efeitos dos fármacosRESUMO
Substituted 2-iminothiolanes (X2ITs) are new heterobifunctional crosslinking agents designed for the preparation of disulfide-linked conjugates with enhanced resistance to reduction. Based upon 2-IT substituted at the 4 and/or 5 position, these reagents appear to function by sterically protecting the conjugate disulfide bond from attack by thiolate nucleophiles. Here, we have used the X2ITs to prepare and evaluate a series of immunoconjugates (antibody-cytotoxin conjugates) between the murine monoclonal antibody 791/T36, which recognizes a 72-kDa surface antigen present on many human tumor cells, and RTA30, the naturally occurring 30-kDa glycoform of ricin A chain. The X2IT-linked conjugates were also compared to immunoconjugates prepared with N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP) and 4-[(succinimidyloxy)carbonyl]-alpha-methyl-alpha-(2- pyridyldithio)toluene (SMPT), as well as with methyl- and dimethyl-substituted structural analogs of SPDP. In vitro, 791-(X2IT)-TNB model compounds exhibited a 6000-fold range of stabilities. In contrast, the corresponding 791-(X2IT)-RTA30 immunoconjugates were up to 20-fold more stable than conjugates made with unhindered linkages. These improvements resulted in immunoconjugates with prolonged serum half-lives in animals. Our data indicate that one of the crosslinking agents, 5-methyl-2-iminothiolane (M2IT), has optimal properties for the preparation of disulfide crosslinked immunoconjugates intended for therapeutic use in that (i) it is highly water soluble and reacts rapidly with protein amino groups at neutral pH, preserving the positive charge, (ii) it forms conjugates with RTA30 efficiently, and (iii) its conjugates exhibited enhanced disulfide bond stability in vitro and in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Reagentes de Ligações Cruzadas , Imunoconjugados/isolamento & purificação , Tiofenos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/farmacocinética , Dissulfetos/química , Dissulfetos/isolamento & purificação , Estabilidade de Medicamentos , Humanos , Imunoconjugados/química , Imunoconjugados/farmacocinética , Imunoglobulina G/química , Imunoglobulina G/isolamento & purificação , Imunotoxinas/química , Imunotoxinas/isolamento & purificação , Imunotoxinas/farmacocinética , Masculino , Camundongos , Modelos Químicos , Ratos , Ratos Sprague-Dawley , Ricina/química , Ricina/isolamento & purificação , Ricina/farmacocinética , Células Tumorais Cultivadas/imunologiaRESUMO
We have used genetic engineering to obtain secretion of anti-human CD5 antibody fragments from Escherichia coli for conjugation to the 30-kDa form of ricin A chain (RTA30). This was accomplished by introducing stop codons at two positions in the hinge region of the human IgG1 gene so that coexpression of the truncated heavy-chain genes (Fd') with a light chain would result in Fab' and/or F(ab')2 proteins containing either one or two interheavy-chain cysteines. An Fd' gene encoding both interheavy-chain cysteines yielded a mixture of F(ab')2 and Fab', which could be separated by size-exclusion chromatography. An Fd' gene encoding only one interheavy-chain cysteine yielded primarily Fab'. Purified F(ab')2 protein was equivalent to unlabeled chimeric IgG in competing for binding of IgG with CD5 antigen, while the molar concentration of the monovalent Fab' required for 50% binding inhibition was 4- to 5-fold higher than IgG. An immunoconjugate was prepared with Fab' by direct coupling to the unique free cysteine on RTA30. The bivalent F(ab')2 was conjugated to RTA30 after derivatization with the crosslinking agent 5-methyl-2-iminothiolane. These immunoconjugates efficiently killed a CD5+ T-cell line and human peripheral blood T cells.
Assuntos
Antígenos CD/imunologia , Fragmentos Fab das Imunoglobulinas/metabolismo , Imunotoxinas/metabolismo , Ricina/metabolismo , Sequência de Aminoácidos , Reações Antígeno-Anticorpo , Sequência de Bases , Ligação Competitiva , Antígenos CD5 , Linhagem Celular , Relação Dose-Resposta a Droga , Escherichia coli/genética , Engenharia Genética , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Imunotoxinas/toxicidade , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Linfócitos T/efeitos dos fármacosRESUMO
We engineered the ribosome inactivating-protein gelonin (Gel) to generate a family of Gel analogs, each with a single unpaired cysteine residue. The cysteine sites coincide with surface-accessible loops in the probable three-dimensional structure of Gel, or with the positions of endogenous cysteine residues. In most cases, enzymatic activity in vitro was unaltered by this modification. The rGel analogs were conjugated via their unpaired cysteine residue to the anti-CD5 antibody H65, or to H65 Fab and F(ab')2. Several rGel analogs formed immunoconjugates that were up to 6-fold more cytotoxic to antigen-bearing cells than those made with linker-modified rGel, whereas others were less potent. In the rat, the in vivo clearance rates of whole antibody conjugates correlated with their relative in vitro disulfide bond stability, and deconjugation to intact antibody and rGel was the predominant clearance mechanism. Fab conjugates to rGel analogs which differed in their in vitro disulfide bond stability had similar serum clearance rates, suggesting that clearance occurs mainly by removal of intact immunoconjugate from the serum, and is less dependent on deconjugation. Our results demonstrate that rGel analogs with a single cysteine at various positions on the solvent exposed surface are produced efficiently in Escherichia coli (>1 g/liter), and that the position of the cysteine greatly influences the potency and stability of the resulting immunoconjugates.
Assuntos
Cisteína , Imunotoxinas/metabolismo , Proteínas de Plantas/imunologia , Inibidores da Síntese de Proteínas/farmacocinética , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/metabolismo , Ensaio de Imunoadsorção Enzimática , Imunoglobulina G/metabolismo , Masculino , Taxa de Depuração Metabólica , Proteínas de Plantas/biossíntese , Engenharia de Proteínas , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/farmacocinética , Proteínas Inativadoras de Ribossomos Tipo 1 , Relação Estrutura-AtividadeRESUMO
Fusion proteins between cell-targeting domains and cytotoxic proteins should be particularly effective therapeutic reagents. We constructed a family of immunofusion proteins linking humanized Fab, F(ab')2, or single chain antibody forms of the H65 antibody (which recognizes the CD5 antigen on the surface of human T cells) with the plant ribosome-inactivating protein gelonin. We reasoned that such an immunofusion would kill human target cells as efficiently as the previously described chemical conjugates of H65 and gelonin (Better M., Bernhard, S. L., Fishwild, D. M., Nolan, P. A., Bauer, R. J., Kung, A. H. C., and Carroll, S. F. (1994) J. Biol. Chem. 269, 9644-9650) if both the recognition and catalytic domains remained active, and a proper linkage between domains could be found. Immunofusion proteins were produced in Escherichia coli as secreted proteins and were recovered directly from the bacterial culture supernatant in an active form. All of the immunofusion proteins were purified by a common process and were tested for cytotoxicity toward antigen-positive human cells. A 20-60-fold range of cytotoxic activity was seen among the fusion family members, and several fusion proteins were identified which are approximately as active as effective chemical conjugates. Based on these constructs, immunofusion avidity and potency can be controlled by appropriate selection of antibody domains and ribosome-inactivating protein.
Assuntos
Antígenos CD/imunologia , Clonagem Molecular/métodos , Escherichia coli/metabolismo , Fragmentos Fab das Imunoglobulinas/biossíntese , Região Variável de Imunoglobulina/biossíntese , Proteínas de Plantas/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Linfócitos T/imunologia , Adulto , Complexo Antígeno-Anticorpo , Antígenos CD5 , Linhagem Celular , Humanos , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Leves de Imunoglobulina/biossíntese , Cadeias kappa de Imunoglobulina/biossíntese , Reação em Cadeia da Polimerase/métodos , Inibidores da Síntese de Proteínas , Proteínas Inativadoras de Ribossomos Tipo 1RESUMO
The pharmacokinetics of several proteins were investigated using two different assays. A 23 kDa recombinant protein fragment of bactericidal/permeability-increasing protein (rBPI23) was radiolabeled with 125I using Iodo-beads and administered rats. Plasma samples were collected and assayed for 125I-rBPI23 by radioactivity. In a separate experiment, rBPI23 was administered to rats and plasma samples were assayed for rBPI23 by ELISA. The clearance determined from plasma concentrations of 125I-rBPI23 measured by radioactivity was about 2.5-fold lower than that of rBPI23 determined by ELISA. In addition, the steady state volumes of distribution and mean residence times of 125I-rBPI23 measured by radioactivity were four-fold and 10-fold greater, respectively, compared to those measured by the ELISA method. By studying several proteins with a range of molecular weights, we found that the pharmacokinetics of proteins below about 60 kDa were different when assayed by radioactivity or ELISA, but those of proteins with molecular weights of at least 80 kDA revealed only minor differences. To determine which assay method yielded the correct plasma pharmacokinetic profile, rBPI23 was metabolically labeled with 35S-methionine and administered to rats, and plasma samples were assayed by radioactivity. The concentration-time profile assessed by this method was very close to that determined by ELISA. Exposing rBPI23 to chloramine-T (the oxidant used in the iodination process) and measuring its plasma concentration by ELISA revealed pharmacokinetics similar to those of the iodinated protein measured by radioactivity. In contrast, radiolabeling rBPI23 using iodinated Bolton-Hunter reagent (which avoids exposing the protein to oxidant), and measuring 125I-rBPI23 by radioactivity, yielded pharmacokinetics that were similar, although not identical, to the pharmacokinetics of rBPI23 measured by ELISA. Thus, our data suggest that directly iodinating low-molecular-weight proteins by oxidation procedures alters their clearance from the blood, preventing reliable determination of pharmacokinetic parameters.
Assuntos
Anti-Infecciosos/farmacocinética , Proteínas Sanguíneas/farmacocinética , Proteínas de Membrana , Animais , Anti-Infecciosos/administração & dosagem , Anti-Infecciosos/sangue , Peptídeos Catiônicos Antimicrobianos , Atividade Bactericida do Sangue , Proteínas Sanguíneas/administração & dosagem , Ensaio de Imunoadsorção Enzimática , Humanos , Injeções Intravenosas , Radioisótopos do Iodo , Marcação por Isótopo , Masculino , Peso Molecular , Oxirredução , Coelhos , Ratos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/sangue , Proteínas Recombinantes/farmacocinéticaRESUMO
Antibody immunoconjugates were made with native and recombinant forms of the type-I ribosome inactivating protein from barley (BRIP) and with three recombinant BRIP (rBRIP) analogs engineered to contain a unique cysteine residue near the C terminus (at amino acid 256, 270, or 277). rBRIP and all three cysteine analogs (rBRIPc256, rBRIPc270, and rBRIPc277) were produced in E. coli, with yields of soluble protein as high as 1 g/L, and were as active as native BRIP in inhibiting protein synthesis in vitro. Interestingly, the position of the engineered cysteine influenced not only the efficiency of conjugation to antibody but also the efficacy and disulfide bond stability of the immunoconjugates. Anti-CD5 antibody conjugates prepared with native and rBRIP were relatively inactive against antigen-positive target cells, while the conjugate made with rBRIPc277 was 5-fold more cytotoxic. Anti-CD7 antibody conjugates made with rBRIPc277 or rBRIPc270 also exhibited improved potency and stability compared to the conjugate with native BRIP. These results indicate that engineering a cysteine residue into selected positions near the C-terminus of a type-IRIP such as BRIP can improve immunoconjugate yield, disulfide bond stability, and potency.