Inhibition of TNF-alpha produced by Kupffer cells protects against the nonspecific liver toxicity of immunotoxin anti-Tac(Fv)-PE38, LMB-2

LMB-2 (anti-Tac(Fv)-PE38) is a recombinant immunotoxin composed of the Fv fragment of the anti-Tac Ab fused to a 38-kDa form of Pseudomonas: exotoxin A. Recent clinical trials showed that LMB-2 is a promising agent for the treatment of patients with Tac-positive leukemia or lymphoma. One major side effect that needs to be overcome is nonspecific liver toxicity. In the current study, we have analyzed the mechanism of this toxicity using a mouse model. Mice that were injected with a lethal dose of LMB-2 showed severe hepatic necrosis. Immunohistochemistry revealed that LMB-2 accumulated in Kupffer cells in the liver, suggesting that the damage to the hepatocytes was indirect. When we examined the effects of LMB-2 on peritoneal macrophages, cells in the same lineage as Kupffer cells, we found that LMB-2 induced the production of TNF-alpha by these cells. Following LMB-2 administration to mice, the levels of TNF-alpha in the liver increased to very high levels, whereas the rise in serum levels was modest. In addition, the LMB-2-induced liver toxicity was blocked by a specific TNF binding protein (TNFsRp55). Liver toxicity was also blocked by indomethacin, which also blocked the rise of TNF-alpha in the liver. Both TNFsRp55 and indomethacin treatment protected mice against a lethal dose of LMB-2. These data indicate that TNF-alpha produced in the liver by Kupffer cells has an important causal role in the nonspecific liver toxicity of LMB-2. These findings have important clinical implications for the use of immunotoxins in the therapy of patients with cancer.     

Mutants of immunotoxin anti-Tac(dsFv)-PE38 with variable number of lysine residues as candidates for site-specific chemical modification. 1. Properties of mutant molecules

Chemical modification of proteins with substances such as poly(ethylene glycol) can add useful properties to proteins. Currently PEGylation is done in a random manner utilizing amino residues dispersed throughout a protein. For proteins such as immunotoxins, which have several different functional domains, random modification leads to inactivation. To determine if we could produce an immunotoxin with a diminished number of lysine residues so that chemical modification could be restricted to certain regions of the protein, we chose the recombinant immunotoxin anti-Tac(dsFv)-PE38 that has 13 lysine residues in the Fv portion and 3 in the toxin. We prepared a series of mutants with 0-12 lysines in the Fv and 0 or 3 in the toxin. Almost all of these molecules retain full biological activity. Our data indicate that replacement of lysine residues can be achieve without loss of biological potency. These molecules are a useful starting point to carry out site-specific PEGylation experiments.     

Mik-beta 1(Fv)-PE40, a recombinant immunotoxin cytotoxic toward cells bearing the beta-chain of the IL-2 receptor.

Mik-beta 1 is a mAb that binds to the beta subunit of the IL-2R. We have constructed a recombinant single chain immunotoxin Mik-beta 1(Fv)-PE40 by genetically fusing the H and L V domains of Mik-beta 1 to each other via a peptide linker, and then to PE40, a derivative of Pseudomonas exotoxin. Mik-beta 1(Fv)-PE40 was selectively cytotoxic for cells expressing high levels of IL-2R beta (p75) subunit. Mik-beta 1(Fv)-PE40 was cytotoxic to the NK cell line YT-S, which expresses p75 but not p55 subunits, with an IC50 of 6 ng/ml. The ATL line HUT-102 was less sensitive, with an IC50 of 200 ng/ml. However, the IC50 could be lowered to 11 ng/ml when Mik-beta 1(Fv)-PE40 was allowed to bind to HUT-102 cells at 4 degrees C for 4 h before overnight incubation at 37 degrees C. An excess of Mik-beta 1 but not of anti-Tac, the anti-p55 mAb, prevented the cytotoxicity of Mik-beta 1(Fv)-PE40. We constructed a more active version of Mik-beta 1(Fv)-PE40, designated Mik-beta 1(Fv)-PE40KDEL, by converting the carboxyl-terminus of the toxin from -REDLK to -KDEL. Mik-beta 1(Fv)-PE40KDEL showed an IC50 of 2 ng/ml toward YT-S cells and 35 ng/ml toward HUT-102 cells. Binding studies using radioiodinated Mik-beta 1 showed that Mik-beta 1(Fv)-PE40 bound to the p75 receptor subunit with 11% of the affinity of the native Mik-beta 1 antibody. Mik-beta 1(Fv)-PE40 may be a useful reagent to study cells that express IL-2R, and it deserves further study as a possible treatment for cancers in which the malignant cells express high numbers of p75 subunit.     

Study of cystinaminopeptidase heterogeneity in animal sera by the isoelectric focusing method.

Study of the quantitative distribution of cystinaminopeptidase activity in the serum of pregnant and control animals (cow, sheep and rat) by the isoelectric focusing method showed heterogeneity of the cystinaminopeptidase activity of the animals' serum enzymatic system, which was well pronounced in the cow and rat and less pronounced in the sheep. The authors assume from their results that the principal difference between the pregnant and control animals is not the total increase in the level of activity of the enzyme in question, but the shift in its incidence, or the increase in activity at a given pH. It is possible that pregnancy, in man and animals, produces in the serum cystinaminopeptidase system (which is evidently heterogeneous even under normal, physiological conditions) given changes, i.e. the formation of new fractions with a different pI from the controls; other fractions disappear, or their activity diminishes.     

Analysis of site-directed mutations in human pro-alpha 2(I) collagen which block cleavage by the C-proteinase

We have used site-directed mutagenesis to obtain human pro alpha 2(I) cDNAs containing novel mutations designed to inhibit cleavage at the C-proteinase site. Deletion of six relatively conserved amino acids which surround the cleavage site did not interfere with assembly of the triple helix in transfected rat cells, but blocked cleavage of the constituent mutated chains by endogenous C-proteinase. Substitution for a conserved Asp, which forms part of the Ala-Asp bond cleaved by C-proteinase, also blocked cleavage by endogenous C-proteinase. The conserved Asp is, therefore, a necessary component of the C-proteinase cleavage site. Incubation in vitro with a purified mouse C-proteinase, confirmed both mutations to be resistant to cleavage by high concentrations of the physiologically relevant enzyme. Mutant pro alpha 2(I) chains, resistant to cleavage by C-proteinase in culture media, were processed in cell layers by a different protease which cleaved telopeptide domains. Naturally occurring mutations at the C-proteinase site have not been described in human patients. The mutations characterized here, further define the C-proteinase cleavage site and provide reagents which may be informative when introduced into transgenic mice.     

Therapeutic Efficacy of Cintredekin Besudotox (IL13-PE38QQR) in Murine Lung Fibrosis Is Unaffected by Immunity to Pseudomonas aeruginosa Exotoxin A

Background

We have previously explored a therapeutic strategy for specifically targeting the profibrotic activity of IL-13 during experimental pulmonary fibrosis using a fusion protein comprised of human IL-13 and a mutated form of Pseudomonas aeruginosa exotoxin A (IL13-PE) and observed that the intranasal delivery of IL13-PE reduced bleomycin-induced pulmonary fibrosis through its elimination of IL-13-responsive cells in the lung. The aim of the present study was to determine whether the presence of an immune response to P. aeruginosa and/or its exotoxin A (PE) would diminish the anti-fibrotic properties of IL13-PE.

Methodology/Principal Findings

Fourteen days after P. aeruginosa infection, C57BL/6 mice were injected with bleomycin via the intratracheal route. Other groups of mice received 4 doses of saline or IL13-PE by either intranasal or intraperitoneal application, and were challenged i.t. with bleomycin 28 days later. At day 21 after bleomycin, all mice received either saline vehicle or IL13-PE by the intranasal route and histopatological analyses of whole lung samples were performed at day 28 after bleomycin. Intrapulmonary P. aeruginosa infection promoted a neutralizing IgG2A and IgA antibody response in BALF and serum. Surprisingly, histological analysis showed that a prior P. aeruginosa infection attenuated the development of bleomycin-induced pulmonary fibrosis, which was modestly further attenuated by the intranasal administration of IL13-PE. Although prior intranasal administration of IL13-PE failed to elicit an antibody response, the systemic administration of IL13-PE induced a strong neutralizing antibody response. However, the prior systemic sensitization of mice with IL13-PE did not inhibit the anti-fibrotic effect of IL13-PE in fibrotic mice.

Conclusions

Thus, IL13-PE therapy in pulmonary fibrosis works regardless of the presence of a humoral immune response to Pseudomonas exotoxin A. Interestingly, a prior infection with P. aeruginosa markedly attenuated the pulmonary fibrotic response suggesting that the immune elicitation by this pathogen exerts anti-fibrotic effects.     

Changing the antigen binding specificity by single point mutations of an anti-p24 (HIV-1) antibody

The murine mAb CB4-1 raised against p24 (HIV-1) recognizes a linear epitope of the HIV-1 capsid protein. Additionally, CB4-1 exhibits cross-reactive binding to epitope-homologous peptides and polyspecific reactions to epitope nonhomologous peptides. Crystal structures demonstrate that the epitope peptide (e-pep) and the nonhomologous peptides adopt different conformations within the binding region of CB4-1. Site-directed mutagenesis of the fragment variable (Fv) region was performed using a single-chain (sc)Fv construct of CB4-1 to analyze binding contributions of single amino acid side chains toward the e-pep and toward one epitope nonhomologous peptide. The mutations of Ab amino acid side chains, which are in direct contact with the Ag, show opposite influences on the binding of the two peptides. Whereas the affinity of the e-pep to the CB4-1 scFv mutant heavy chain variable region Tyr(32)Ala is decreased 250-fold, the binding of the nonhomologous peptide remains unchanged. In contrast, the mutation light chain variable region Phe(94)Ala reduces the affinity of the nonhomologous peptide 10-fold more than it does for the e-pep. Thus, substantial changes in the specificity can be observed by single amino acid exchanges. Further characterization of the scFv mutants by substitutional analysis of the peptides demonstrates that the effect of a mutation is not restricted to contact residues. This method also reveals an inverse compensatory amino acid exchange for the nonhomologous peptide which increases the affinity to the scFv mutant light chain variable region Phe(94)Ala up to the level of the e-pep affinity to the wild-type scFv.     

Reduction of the Pa(CO2) set point during hyperthermic exercise in the sheep

In animals that rely on the respiratory system for both gas exchange and heat loss, exercise can generate conflict between chemoregulation and thermoregulation. We hypothesized that in panting animals, hypocapnia during hyperthermic exercise reflects a reduction in the arterial CO2 tension (Pa(CO2)) set point. To test this hypothesis, five sheep were subjected to tracheal insufflations of CO2 or air (control) at 3-4 L min(-1) in 3 min bouts at 5 min intervals over 31 min of exercise. During exercise, rectal temperature and minute ventilation (V(E)) rose continuously while Pa(CO2) fell from 35.4+/-3.1 to 18.6+/-2.9 Torr and 34.3+/-2.4 to 18.7+/-1.5 Torr in air and CO2 trials, respectively. Air insufflations did not affect V(E) or Pa(CO2). V(E) increased during CO2 insufflations via a shift to higher tidal volume and lower frequency. CO2 insufflations also increased Pa(CO2), although not above the pre-exercise level. Within 5 min after each CO2 insufflation, Pa(CO2) had decreased to match that following the equivalent air insufflation. These results are consistent with a reduced Pa(CO2) set point or an increased gain of the Pa(CO2) regulatory system during hyperthermic exercise. Either change in the control of Pa(CO2) could facilitate respiratory evaporative heat loss by mitigating homeostatic conflict.     

De novo design and Rosetta-based assessment of high-affinity antibody variable regions (Fv) against the SARS-CoV-2 spike receptor binding domain (RBD)

The continued emergence of new SARS-CoV-2 variants has accentuated the growing need for fast and reliable methods for the design of potentially neutralizing antibodies (Abs) to counter immune evasion by the virus. Here, we report on the de novo computational design of high-affinity Ab variable regions (Fv) through the recombination of VDJ genes targeting the most solvent-exposed hACE2-binding residues of the SARS-CoV-2 spike receptor binding domain (RBD) protein using the software tool OptMAVEn-2.0. Subsequently, we carried out computational affinity maturation of the designed variable regions through amino acid substitutions for improved binding with the target epitope. Immunogenicity of designs was restricted by preferring designs that match sequences from a 9-mer library of “human Abs” based on a human string content score. We generated 106 different antibody designs and reported in detail on the top five that trade-off the greatest computational binding affinity for the RBD with human string content scores. We further describe computational evaluation of the top five designs produced by OptMAVEn-2.0 using a Rosetta-based approach. We used Rosetta SnugDock for local docking of the designs to evaluate their potential to bind the spike RBD and performed “forward folding” with DeepAb to assess their potential to fold into the designed structures. Ultimately, our results identified one designed Ab variable region, P1.D1, as a particularly promising candidate for experimental testing. This effort puts forth a computational workflow for the de novo design and evaluation of Abs that can quickly be adapted to target spike epitopes of emerging SARS-CoV-2 variants or other antigenic targets.     

Reduction in the toxicity of aminopterin—monoclonal-antibody conjugates by leucovorin

Although aminopterin(AMN)-antibody drug conjugates have been demonstrated to have a greatly increased antitumour efficacy compared to the free drug, their use is limited by an increase in systemic toxicity manifested by weight loss and bone marrow suppression. Using a murine thymoma model (E3) in inbred mice, the toxicity of a sublethal dose of free AMN could be prevented by the administration of leucovorin 24 h following drug treatment, whilst maintaining the antitumour effect of the drug. The same rescue protocol completely abrogated the antitumour efficacy of AMN-antibody, although toxicity was also diminished. However, the later administration of leucovorin 48–72 h following a sublethal dose of AMN-antibody conjugates resulted in a maintenance of the antitumour efficacy of the immunoconjugates and a reduction in toxicity, with a mean percentage change in mouse weight not significantly different from that of the controls. These studies demonstrate that reversal of toxicity caused by AMN-antibody conjugates can be achieved by leucovorin while maintaining a powerful antitumour effect provided that the dose of leucovorin is administered 48–72 h after the conjugate.     

Evaluation of the ligase chain reaction (LCR) for the detection of point mutations.

The ligase chain reaction (LCR) was evaluated as an amplification method for an in vivo mutation assay. Specifically, the ligase was tested for its ability to selectively amplify a DNA sequence mutated at a single base, in the presence of an excess of wild-type DNA. As a model template a 370-bp DNA fragment of the mouse Ha-ras protooncogene containing an A to T mutation at the second position of codon 61 was used. With the commercially available ligase Ampligase (Epicenter), 250 molecules of mutant fragments could be detected by an enzyme-linked immunoassay with digoxigenin marker (giving a theoretical detection limit of 1 target gene per 10(4) copies of genome). In the analysis of mixtures with corresponding wild-type DNA fragments, a 1:1 mixture resulted in a clearly stronger signal than control samples lacking wild-type and mutant DNA. However, the signal obtained from a 100-fold dilution of the mutant DNA with wild-type DNA could not be distinguished from the background noise. In this particular form, LCR lacks sufficient selectivity to be applied to an in vivo situation, where the ratio of mutant to wild-type DNA sequences might be expected to lie around 1:10(6).     

Reduction of BCNU toxicity to lung cells by high-level expression of O(6)-methylguanine-DNA methyltransferase

1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) is an important cause of pulmonary toxicity. BCNU alkylates DNA at the O(6) position of guanine. O(6)-methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that removes alkyl groups from the O(6) position of guanine. To determine whether overexpression of MGMT in a lung cell reduces BCNU toxicity, the MGMT gene was transfected into A549 cells, a lung epithelial cell line. Transfected A549 cell populations demonstrated high levels of MGMT RNA, MGMT protein, and DNA repair activity. The overexpression of MGMT in lung epithelial cells provided protection from the cytotoxic effects of BCNU. Control A549 cells incubated with 100 microM BCNU had a cell survival rate of 12.5 +/- 1.2%; however, A549 cells overexpressing MGMT had a survival rate of 71.8 +/- 2.7% (P < 0.001). We also demonstrated successful transfection of MGMT into human pulmonary artery endothelial cells and a primary culture of rat type II alveolar epithelial cells with overexpression of MGMT, resulting in significant protection from BCNU toxicity. These data suggest that overexpression of DNA repair proteins such as MGMT in lung cells may protect the lung cells from cytotoxic effects of cancer chemotherapy drugs such as BCNU.     

Pleiotrophic effects of point mutations in yeast tRNA(Asp) on the base modification pattern.

The base-modification pattern has been studied in several synthetic variants of yeast tRNA(Asp) injected into Xenopus laevis oocytes. Certain point mutations in the D-stem and the variable loop of the tRNA led to considerably decreased levels of m1G37, psi 40 and Q34/manQ34 in the anticodon stem or loop and an increased rate of synthesis for m5C49 in the T-stem. The formation of m2G6 in the aminoacyl-stem was not affected in any of the tRNA-variants. Thus, mutations in one part of the tRNA-molecule can have long-range effects on the interactions between another part of the tRNA and the tRNA modifying enzymes.     

Reduction of ENU-induced transversion mutations by the isoflavone genistein in Escherichia coli

In studies of mutagenesis induced by the carcinogen N-ethyl-N-nitrosourea (ENU) in the bacterium Escherichia coli FX-11, it was observed that G:C to A:T transitions did not require the inducible umuDC gene products, while a portion of the A:T to G:C transitions and all transversion mutations were dependent on a functional umuC gene. This observation suggested that the different base substitutions may result from differential processing of specific DNA adducts produced by ENU. To further understand these processes, we have investigated the effect of the soybean isoflavone genistein on the production of ENU-induced mutations. This compound, in particular, has been shown to exhibit numerous effects including the inhibition of the growth or proliferation of a variety of cancers, inhibition of angiogenesis, inhibition of tyrosine protein kinases and anti-oxidant properties. In our experiments, tyrosine defective (TyrA(-)) E. coli were exposed to ENU and a portion of the ENU-treated cells were exposed to genistein. The results showed a three-fold reduction in the overall mutation frequency when cells were treated with genistein subsequent to ENU-exposure and this anti-mutagenic effect was dependent on the dose of genistein employed. However, only certain types of base substitution mutagenesis were affected. In particular, transversion mutations were reduced an average of about 8.5-fold, while transitions were not greatly affected. In addition, UV-mutagenesis was reduced about three-fold and induction of the SOS response (as monitored with a sulA-lacZ fusion) was decreased. These results suggest that genistein may interfere with expression of the SOS response, including the UmuC-mediated lesion bypass mechanism that is necessary for UV-mutagenesis and the generation of transversions by ENU in E. coli.     

Mapping and validation of chromosome regions conferring boron toxicity tolerance in wheat (Triticum aestivum)

Boron is an essential plant micro-nutrient which can be phytotoxic to plants if present in soils in high concentration. Boron toxicity has been recognised as an important problem limiting production in the low rainfall areas of southern Australia, West Asia and North Africa. Genetic variation for boron toxicity tolerance in wheat has been well-characterised. The efficiency of breeding for boron toxicity tolerance could be greatly enhanced by the development of molecular markers associated with QTLs for tolerance in wheat. A population of 161 doubled haploids from a cross between the tolerant cultivar Halberd and the moderately sensitive cultivar Cranbrook was used to identify chromosomal regions involved in boron tolerance. A combined RFLP and AFLP linkage map of the Cranbrook x Halberd population was used to identify chromosomal regions involved in the boron tolerance traits measured. Regions on chromosome 7B and 7D were associated with leaf symptom expression. The region on chromosome 7B was also associated with the control of boron uptake and with a reduction in the effect of boron toxicity on root-growth suppression. RFLP markers at the chromosome 7B and 7D loci were shown to be effective in selecting for improved boron tolerance in an alternative genetic background. Halberd alleles at the chromosome 7B locus were associated with the concentration of boron in whole shoots and grain. The concentration of boron in whole shoots and in grain were both related to grain yield in a field trial conducted on soil containing toxic levels of boron. Implications relating to marker-assisted selection for boron toxicity tolerance in wheat are discussed. Received: 3 September 1999 / Accepted: 12 February 2000     

Induction of point mutations by different chemical mechanisms in the liver microsomal assay

A selection of chemical agents with different mechanisms of chemical mutability was tested with the liver microsomal assay by using different bacterial tester strains, namely: Salmonella typhimurium TA 1535, TA 1536, TA 1537, TA 1538 and G46. The tested agents had been selected according to the following criteria. They are all well-known mutagens and can be divided into alkylating agents, anti-metabolites, acridines and those that form radicals in the cell. The mutagens were: dimethylnitrosamine (DMN), diethylnitrosamine (DEN), methyl-nitro-nitrosoguanidine (MNNG), cyclophosphamide, Captan, amethopterine, azathioprine, 6-mecaptopurine, trypaflavine, isoniazide and hydrazine. All except amethopterine gave positive results, showing that this system is very sensitive.In a comparison of the different strains and mutagens we found a correlation between the diameter of the molecule and the permeability of the bacterial cell membrane.     

Detection of point mutations in the chloroplast genome by single-stranded conformation polymorphism analysis

Single-stranded conformation polymorphism (SSCP) analysis was used to examine the mutations of the chloroplast 16S rRNA locus of streptomycin-resistant mutants in Nicotiana plumbaginifolia. DNA fragments of 121, 517, 968 and 1578 bp, each possessing a known point mutation, were generated by polymerase chain reaction (PCR). The resulting fragments were denatured and separated by nondenaturing polyacrylamide gel electrophoresis. Compared to the patterns of the wild-type DNA fragments, the bands of the single-stranded DNA fragments of 121 and 517 bp with base changes were shifted. However, no pattern variations were detected from the DNA fragments of 968 and 1578 bp generated from both wild-type and mutants.