Preparation Of Immunotoxins Research Articles

Story of Pore-Forming Proteins from Deadly Disease-Causing Agents to Modern Applications with Evolutionary Significance.

Animal venoms are a complex mixture of highly specialized toxic molecules. Among them, pore-forming proteins (PFPs) or toxins (PFTs) are one of the major disease-causing toxic elements. The ability of the PFPs in defense and toxicity through pore formation on the host cell surface makes them unique among the toxin proteins. These features made them attractive for academic and research purposes for years in the areas of microbiology as well as structural biology. All the PFPs share a common mechanism of action for the attack of host cells and pore formation in which the selected pore-forming motifs of the host cell membrane-bound protein molecules drive to the lipid bilayer of the cell membrane and eventually produces water-filled pores. But surprisingly their sequence similarity is very poor. Their existence can be seen both in a soluble state and also in transmembrane complexes in the cell membrane. PFPs are prevalent toxic factors that are predominately produced by all kingdoms of life such as virulence bacteria, nematodes, fungi, protozoan parasites, frogs, plants, and also from higher organisms. Nowadays, multiple approaches to applications of PFPs have been conducted by researchers both in basic as well as applied biological research. Although PFPs are very devastating for human health nowadays researchers have been successful in making these toxic proteins into therapeutics through the preparation of immunotoxins. We have discussed the structural, and functional mechanism of action, evolutionary significance through dendrogram, domain organization, and practical applications for various approaches. This review aims to emphasize the PFTs to summarize toxic proteins together for basic knowledge as well as to highlight the current challenges, and literature gap along with the perspective of promising biotechnological applications for their future research.

A Single-Domain Antibody-Based Anti-PSMA Recombinant Immunotoxin Exhibits Specificity and Efficacy for Prostate Cancer Therapy.

Prostate cancer (PCa) is the second most common cancer in men, causing more than 300,000 deaths every year worldwide. Due to their superior cell-killing ability and the relative simplicity of their preparation, immunotoxin molecules have great potential in the clinical treatment of cancer, and several such molecules have been approved for clinical application. In this study, we adopted a relatively simple strategy based on a single-domain antibody (sdAb) and an improved Pseudomonas exotoxin A (PE) toxin (PE24X7) to prepare a safer immunotoxin against prostate-specific membrane antigen (PSMA) for PCa treatment. The designed anti-PSMA immunotoxin, JVM-PE24X7, was conveniently prepared in its soluble form in an Escherichia coli (E. coli) system, avoiding the complex renaturation process needed for immunotoxin preparation by the conventional strategy. The product was very stable and showed a very strong ability to bind the PSMA receptor. Cytotoxicity assays showed that this molecule at a very low concentration could kill PSMA-positive PCa cells, with an EC50 value (concentration at which the cell viability decreased by 50%) of 15.3 pM against PSMA-positive LNCaP cells. Moreover, this molecule showed very good killing selectivity between PSMA-positive and PSMA-negative cells, with a selection ratio of more than 300-fold. Animal studies showed that this molecule at a very low dosage (5 × 0.5 mg/kg once every three days) completely inhibited the growth of PCa tumors, and the maximum tolerable dose (MTD) was more than 15 mg/kg, indicating its very potent tumor-treatment ability and a wide therapeutic window. Use of the new PE toxin, PE24X7, as the effector moiety significantly reduced off-target toxicity and improved the therapeutic window of the immunotoxin. The above results demonstrate that the designed anti-PSMA immunotoxin, JVM-PE24X7, has good application value for the treatment of PCa.

Preparation of a novel EGFR specific immunotoxin and its efficacy of anti-colorectal cancer in vitro and in vivo.

Epithelial growth factor receptor (EGFR), as a malignancy marker, is overly expressed in multiple solid tumors including colorectal neoplasms, one of the most prevalent malignancies worldwide. The main objective of this study is to enhance the efficacy of anti-tumor therapy targeting EGFR by constructing a novel EGFR-specific immunotoxin (C-CUS245C) based on Cetuximab and recombinant Cucurmosin (CUS245C). E. coli BL21 (DE3) PlysS (E. coli) was used to express CUS245C with a cysteine residue inserting to the C-terminus of Cucurmosin. Then immobilized metal ion affinity chromatography (IMAC) was used to purify CUS245C. The chemical conjugation method was used for the preparation of C-CUS245C. Then dialysis and IMAC were used to purify C-CUS245C. Western blot as well as SDS-PAGE was carried out to characterize the formation of C-CUS245C. At last the anti-colorectal cancer activity of C-CUS245C was investigated in vitro and in vivo. CUS245C with high purity could be obtained from the prokaryotic system. C-CUS245C was successfully constructed and highly purified. The cytotoxicity assays in vitro showed a significant proliferation inhibition of C-CUS245C on EGFR-positive cells for 120 h with IC50 values less than 0.1 pM. Besides, the anti-tumor efficacy of C-CUS245C was remarkably more potent than that of Cetuximab, CUS245C, and C + CUS245C (P < 0.001). Whereas the cytotoxicity of C-CUS245C could hardly be detected on EGFR-null cell line. Our results also showed that C-CUS245C had efficacy of anti-colorectal cancer in mouse xenograft model, indicating the therapeutic potential of C-CUS245C for the targeted therapy of colorectal neoplasms. C-CUS245C exhibits potent and EGFR-specific cytotoxicity. Insertional mutagenesis technique is worthy to be adopted in the preparation of immunotoxin. Immunotoxin can be highly purified through dialysis followed by IMAC.

ADP-ribosylating bacterial toxins : Pseudomonas exotoxin A

It is well known that a number of toxins produced by bacteria exert their action by ADP-ribosylating reaction to certain proteins which are essential for normal eukaryotic cellular functions. Most of these toxins are composed of two moieties, A and B. The B moiety mediates the binding to the specific receptor on the surface of toxin-sensitive cells, while the A moiety is responsible for the enzymatic ADP-ribosylating activity. Pseudomonas exotoxin A (PEA) is the most toxic component of the extracellular products produced by Pseudomonas aeruginosa. The three domain model of PEA has been well established : domain I, domain II, and domain III exerting binding, translocation, and ADP-ribosylating activities, respectively. Because of the cytotoxic ADP-ribosylating nature of PEA, it has been suggested as a good candidate in the preparation of immunotoxins. In this minireview article, we discuss the structure and function of the bacterial ADP-ribosylating toxins including PEA and compare the differences particularly between PEA and other valevant toxins.

High in Vitro Anti-Tumor Efficacy of Dimeric Rituximab/Saporin-S6 Immunotoxin.

The anti-CD20 mAb Rituximab has revolutionized lymphoma therapy, in spite of a number of unresponsive or relapsing patients. Immunotoxins, consisting of toxins coupled to antibodies, are being investigated for their potential ability to augment Rituximab efficacy. Here, we compare the anti-tumor effect of high- and low-molecular-weight Rituximab/saporin-S6 immunotoxins, named HMW-IT and LMW-IT, respectively. Saporin-S6 is a potent and stable plant enzyme belonging to ribosome-inactivating proteins that causes protein synthesis arrest and consequent cell death. Saporin-S6 was conjugated to Rituximab through an artificial disulfide bond. The inhibitory activity of HMW-IT and LMW-IT was evaluated on cell-free protein synthesis and in two CD20+ lymphoma cell lines, Raji and D430B. Two different conjugates were separated on the basis of their molecular weight and further characterized. Both HMW-IT (dimeric) and LMW-IT (monomeric) maintained a high level of enzymatic activity in a cell-free system. HMW-IT, thanks to a higher toxin payload and more efficient antigen capping, showed stronger in vitro anti-tumor efficacy than LMW-IT against lymphoma cells. Dimeric HMW-IT can be used for lymphoma therapy at least for ex vivo treatments. The possibility of using HMW-IT augments the yield in immunotoxin preparation and allows the targeting of antigens with low internalization rates.

Application of Sodium Dodecyl Sulfate-Capillary Gel Electrophoresis to the Characterization of Ricin A-Chain Immunotoxins

This study was undertaken to use sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) for the characterization of immunotoxins constructed by cross-linking ricin A-chain (RTA) to monoclonal antibody (MAb). SDS-CGE could separate mono- and di-RTA-conjugated MAbs from unreacted MAb, as identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Resolution of RTA-MAb conjugates was improved by decreasing the running voltage from 15 to 5 kV. SDS-CGE was also useful for characterizing aggregates of RTA or deglycosylated RTA (dgRTA) that was formed during immunotoxin preparation. Of the RTA isomers with different carbohydrate contents, RTA1, which has low carbohydrate content, was found to be more susceptible to aggregation than RTA2. In the SDS-CGE of dgRTA, both covalently and non-covalently linked aggregates were observed under reducing and non-reducing conditions. The findings of this study suggest that SDS-CGE can be used to optimize methods of preparing effective immunotoxins and for checking degradants and aggregates.

Preparation of immunotoxin using granulocyte colony-stimulating factor (G-CSF), for cancer therapy

Preparation of immunotoxin using granulocyte colony-stimulating factor (G-CSF), for cancer therapy

Bacterial expression of biologically active recombinant musarmin 1 from bulbs of Muscari armeniacum L. and Miller

Musarmins are type 1 ribosome-inactivating proteins with N-glycosidase activity on the 28 S rRNA that are present in bulbs of Muscari armeniacum L. and Miller at rather low concentrations. In the present work, a cDNA fragment coding for musarmin 1 was sub-cloned and expressed in Escherichia coli. The recombinant protein (rMU1) was synthesised as a polypeptide of 295 amino acids that was delivered to the periplasm and processed. Recombinant musarmin 1 present in the periplam has two forms: insoluble with a molecular mass of 29,423 and soluble with a molecular mass of 29,117 because of a small proteolytic shortening with respect to the insoluble one, presumably in the C-terminal. The yield of protein homogeneous by polyacrylamide gel electrophoresis was 23 mg l −1 of bacterial culture. The recombinant musarmin 1 forms isolated from both the soluble and the insoluble (upon refolding) fractions retained full translational inhibitory and 28 S rRNA N-glycosidase activities as compared with the native protein. The recombinant protein displayed great stability towards trypsin, collagenase, rat plasma and rat liver protein extract, but was sensitive to the action of papain and proteinase K. The easy availability and full activity of the recombinant musarmin 1 makes it a good candidate for the preparation of immunotoxins for targeted therapy and for the construction of transgenic plants expressing it as antipathogenic agent.

Chemical construction of immunotoxins.

Immunotoxins are chimeric proteins consisting of an antibody linked to a toxin. The antibodies most frequently used for the preparation of immunotoxins are murine monoclonal antibodies belonging to IgG isotype. The most used toxins for the chemical construction of immunotoxins are Ricin toxin A chain in its deglycosylated form and recombinant Pseudomonas endotoxin with the cell-binding domain deleted. The linkage of the antibody to the toxin can be accomplished by chemical methods using reagents that crosslink antibody to toxin. The usual crosslinkers attach disulfide groups into the antibody molecule to form a disulfide bond between the antibody and the toxin. Disulfide bonds are susceptible to reduction in the cytoplasm of the targeted cells thereby releasing the toxin so that it can exert its cytotoxic activity only into the cells (e.g., tumor cells) binding the antibody moiety. This article describes various methods to obtain antibodies and toxins and several procedures for their crosslinking as well as "in vitro" and "in vivo" testing of the immunotoxins efficacy.

Purification, characterization and molecular cloning of trichoanguin, a novel type I ribosome-inactivating protein from the seeds of Trichosanthes anguina

The seeds of the plant Trichosanthes anguina contain a type I ribosome-inactivating protein (RIP), designated trichoanguin, which was purified to apparent homogeneity by the combined use of ion-exchange chromatographies, i.e. first with DE-52 cellulose and then with CM-52 cellulose. The protein was found to be a glycoprotein with a molecular mass of 35 kDa and a pI of 9.1. It strongly inhibits the protein synthesis of rabbit reticulocyte lysate, with an IC50 of 0.08 nM, but only weakly that of HeLa cells, with an IC50 of 6 µM. Trichoanguin cleaves at the A4324 site of rat 28 S rRNA by its N-glycosidase activity. The cDNA of trichoanguin consists of 1039 nt and encodes an open reading frame coding for a polypeptide of 294 amino acid residues. The first 19 residues of this polypeptide encode a signal peptide sequence and the last 30 residues comprise an extension at its C-terminus. There are four potential glycosylation sites, located at Asn-51, Asn-65, Asn-201 and Asn-226. A comparison of the amino acid sequence of trichoanguin with those of RIPs such as trichosanthin, α-momorcharin, ricin A-chain and abrin A-chain reveals 55%, 48%, 36% and 34% identity respectively. Molecular homology modelling of trichoanguin indicates that its tertiary structure closely resembles those of trichosanthin and α-momorcharin. The large structural similarities might account for their common biological effects such as an abortifacient, an anti-tumour agent and anti-HIV-1 activities. Trichoanguin contains two cysteine residues, Cys-32 and Cys-155, with the former being likely to be located on the protein surface, which is directly amenable for conjugation with antibodies to form immunoconjugates. It is therefore conceivable that trichoanguin might be a better type I RIP than any other so far examined for the preparation of immunotoxins, with a great potential for application as an effective chemotherapeutic agent for the treatment of cancer.

Purification, characterization and molecular cloning of trichoanguin, a novel type I ribosome-inactivating protein from the seeds of Trichosanthes anguina

The seeds of the plant Trichosanthes anguina contain a type I ribosome-inactivating protein (RIP), designated trichoanguin, which was purified to apparent homogeneity by the combined use of ion-exchange chromatographies, i.e. first with DE-52 cellulose and then with CM-52 cellulose. The protein was found to be a glycoprotein with a molecular mass of 35 kDa and a pI of 9.1. It strongly inhibits the protein synthesis of rabbit reticulocyte lysate, with an IC50 of 0.08 nM, but only weakly that of HeLa cells, with an IC50 of 6 microM. Trichoanguin cleaves at the A4324 site of rat 28 S rRNA by its N-glycosidase activity. The cDNA of trichoanguin consists of 1039 nt and encodes an open reading frame coding for a polypeptide of 294 amino acid residues. The first 19 residues of this polypeptide encode a signal peptide sequence and the last 30 residues comprise an extension at its C-terminus. There are four potential glycosylation sites, located at Asn-51, Asn-65, Asn-201 and Asn-226. A comparison of the amino acid sequence of trichoanguin with those of RIPs such as trichosanthin, alpha-momorcharin, ricin A-chain and abrin A-chain reveals 55%, 48%, 36% and 34% identity respectively. Molecular homology modelling of trichoanguin indicates that its tertiary structure closely resembles those of trichosanthin and alpha-momorcharin. The large structural similarities might account for their common biological effects such as an abortifacient, an anti-tumour agent and anti-HIV-1 activities. Trichoanguin contains two cysteine residues, Cys-32 and Cys-155, with the former being likely to be located on the protein surface, which is directly amenable for conjugation with antibodies to form immunoconjugates. It is therefore conceivable that trichoanguin might be a better type I RIP than any other so far examined for the preparation of immunotoxins, with a great potential for application as an effective chemotherapeutic agent for the treatment of cancer.

Probing the Domain Structure of Abrin‐a by Tryptic Digestion

Abrin-a is a potent plant toxin that consists of A and B chains linked by a disulfide bond. The abrin-a A chain (AaTA) has N-glycosidase activity while the abrin-a B chain (AaTB) has galactose-binding activity. By partial tryptic digestion, the domain structure of abrin-a was investigated. Seven tryptic fragments with molecular masses greater than 3500 Da were isolated and characterized. One fragment, designated T-21 and consisting of 153 amino acid residues, contained the major part of the second domain of AaTB and, after cross-linking of T-21 with glutaraldehyde, the reaction product had the same level of hemagglutinating activity as native abrin. When the T-21 fragment was conjugated with AaTA, the conjugate inhibited protein biosynthesis in HeLa cells. This suggests that the T-21 fragment is able to bind specifically to cells; its conjugate facilitates membrane translocation of AaTA into cells and consequently inhibits protein biosynthesis. T-21, with a molecular mass less than AaTB, is therefore a potentially useful substance for the preparation of immunotoxins.

DNA-nuclease activity of the single-chain ribosome-inactivating proteins dianthin 30, saporin 6 and gelonin

The single-chain ribosome-inactivating proteins (sc-RIPs) from plant origin are antiviral and antiproliferative agents employed in the preparation of immunotoxins. Similarly to the A-chains of ricin, sc-RIPs act as rRNA N-glycosidases. We demonstrate here that dianthin 30, saporin 6 and gelonin exert a specific nuclease activity on supercoiled DNA. Four specific sites of cleavage introduced by dianthin 30 and by saporin 6 and two specific sites of cleavage introduced by gelonin have been identified and mapped in pBR322.

Purification and properties of immunotoxins containing one vs. two deglycosylated ricin A chains

A method for the preparation of immunotoxins (ITs) containing one and two molecules of deglycosylated ricin A chain (dgA) bound to each molecule of anti-human CD22 mouse IgG1 monoclonal antibody is described. The cross-linking of antibody and dgA molecules was accomplished with N-succinimidyl-oxycarbonyl- α-methyl-(2-pyridyldithio). The two ITs were purified by successive chromatographies on Blue-Sepharose, Sephacryl S-200, Blue-Sepharose with NaCl gradient and Sephacryl S-300. The IT with two dgAs was seven-fold more cytotoxic to CD22 + Daudi cells than that containing one molecule of dgA.

The GLP large scale preparation of immunotoxins containing deglycosylated ricin A chain and a hindered disulfide bond

The large scale preparation of two second generation immunotoxins containing murine monoclonal antibodies and deglycosylated ricin A chain is described. The procedure for the preparation of immunotoxins consists of the derivatization of antibody with SMPT and reduction of dgA with DTT followed by their reaction to establish a hindered interchain disulfide bond. The purification of the immunotoxin includes affinity chromatography on Blue-Sepharose to remove the free antibody and gel filtration on Sephacryl S-200HR to remove any high molecular weight material and free dgA. The two immunotoxins were prepared by GLP procedures and tested for yield, composition, purity, sterility and biological activity.

Production of a pokeweed antiviral protein (PAP)-containing immunotoxin, B43-PAP, directed against the CD19 human B lineage lymphoid differentiation antigen in highly purified form for human clinical trials

We describe a standardized method for the preparation and purification of a potent immunotoxin against B-lineage leukemia/lymphoma cells, constructed with the ribosome inhibitory single chain plant toxin pokeweed antiviral protein (PAP) and a murine IgG1 monoclonal antibody (MoAb) specific for the human B lineage differentiation antigen CD19 for human clinical trials. PAP was prepared from spring leaves of Phytolacca americana plants by ammonium sulfate precipitation and purified to homogeneity by successive steps of ion exchange chromatography. B43 MoAb was produced in vitro by hollow fiber technology and purified to homogeneity by affinity chromatography. PAP toxin and B43 MoAb were modified via their free amino groups prior to their intermolecular conjugation. 2-iminothiolane was used to introduce reactive sulfhydryl groups into PAP and N-succinimidyl 3-(2-pyridyldithio) propionate was used to introduce 2-pyridyl disulfide bonds into B43 MoAb. Modified PAP was reacted with modified B43 MoAb resulting in a sulfhydryl-disulfide exchange reaction and yielding disulfide linked PAP-B43 MoAb conjugates, which we refer to as B43-PAP immunotoxin. B43-PAP immunotoxin was subjected to preparative gel filtration chromotography and cation exchange chromatography to obtain a highly purified, sterile, and pyrogen-free immunotoxin preparation with <5% free antibody contamination and <0.5% free PAP contamination. The final product displayed a high affinity for and a very potent anti-leukemic activity against B lineage leukemia cells. With slight modifications, the procedures detailed in this report should be generally applicable to preparation of other PAP-MoAb conjugates for treatment of cancer or AIDS.

Purification and properties of a new ribosome-inactivation protein with RNA N-glycosidase activity suitable for immunotoxin preparation from the seeds of Momordica cochinchinensis

A ribosome-inactivating protein similar to those already known (Stirpe and Barbieri (1986) FEBS Lett. 195, 1–8) was purified from the seeds of Momordica cochinchinensis. This protein, for which the name of momorcochin-S is proposed, is a glycoprotein, has an M r of approx. 30 000, and an alkaline isoelectric point and can be considered as an iso-form of the previously purified momorcochin from the roots of M. cochinchinensis. Momorcochin-S inhibits protein synthesis by a rabbit-reticulocyte lysate and phenylalanine polymerization by isolated ribosomes, and alters rRNA in a similar manner as the A-chain of ricin and related toxins (Endo et al. (1987) J. Biol. Chem. 262, 5908–5912). Momorcochin-S was linked to a monoclonal antibody (8A) against human plasma cells, and the resulting immunotoxin was selectively toxic to target cells.

Selective killing of human monocytes by an immunotoxin containing partially denatured mistletoe lectin I

The in vitro activity of an immunotoxin (IT) prepared by conjugating the monocyte specific RoMo-1 monoclonal antibody and partially inactivated Mistletoe lectin I (dMLI) containing inactivated B chain has been initially characterized. It is shown that while not affecting other mononuclear cells viability, this IT is capable of selectively destroying human monocytes after 24 h exposure thus resulting in the abrogation of monocyte support for PHA reactivity in mononuclear cell preparations. Therefore it seems to be possible to use this non-sugar binding holotoxin for immunotoxin preparation.

Trichokirin, a ribosome-inactivating protein from the seeds of Trichosanthes kirilowii Maximowicz. Purification, partial characterization and use for preparation of immunotoxins.

A protein, here named trichokirin, was extracted from the seeds of Trichosanthes kirilowii and purified by ion-exchange and gel-filtration chromatography. Trichokirin is a basic glycoprotein of apparent relative molecular mass of 27,000 with a strong ribosome-inactivating activity. Alignment of the trichokirin, trichosanthin and momordin N-terminal sequences shows a substantial degree of homology. Trichokirin was conjugated to a monoclonal antibody directed against the Thy 1.2 antigen with the cleavable dimethyl 3,3'-dithiobispropionimidate cross-linking reagent. This immunotoxin selectively killed leukemia cells expressing the Thy 1.2 antigen. The addition of ammonium chloride, which increases the cytotoxicity of ricin A-chain immunotoxins, blocks that of the trichokirin immunotoxin, suggesting that they enter cells by different mechanisms. In vivo studies showed that the pharmacokinetic properties of the trichokirin immunotoxin could be more advantageous than those of the ricin A-chain immunotoxins for in vivo applications.

Development of a bifunctional crosslinking agent with potential for the preparation of immunotoxins

A new protein crosslinking agent, 2,3-dibromopropionyl-N-hydroxysuccinimide ester, has been synthesized and characterized. The potential use of this compound as a temperature-controllable heterobifunctional crosslinking agent has been investigated using model systems and its reactivity compared with that of chlorambucil-N-hydroxysuccinimide ester. The coupling of 14C-labeled phenylethylamine to lysozyme has been used to illustrate the feasibility of the use of this crosslinking agent for the synthesis of immunotoxins.