Treatment of hepatocellular carcinoma in a mouse xenograft model with an immunotoxin which is engineered to eliminate vascular leak syndrome

Vascular leak syndrome (VLS) is the major dose-limiting toxicity of immunotoxin and interleukin-2 therapy. It has been evidenced that VLS-inducing molecules share a three-amino acid consensus motif, (x)D(y), which may be responsible for initiating VLS. Here we have constructed a recombinant immunotoxin (SMFv-PE38KDEL) by genetically fusing PE38KDEL to a single-chain antibody derived from SM5-1 monoclonal antibody, which has a high specificity for melanoma, hepatocellular carcinoma and breast cancer. In order to eliminate VLS induced by this PE38KDEL-based immunotoxin, a panel of mutants were generated by changing amino acid residues adjacent to its three (x)D(y) motifs in the three-dimensional structure. One of the SMFv-PE38KDEL mutants, denoted as mut1, displayed a similar protein synthesis inhibitory in a reticulocyte lysate translation assay compared to the wild-type SMFv-PE38KDEL (wt). The in vitro cytotoxicity assay indicated that mut1 specifically killed SM5-1 binding protein-positive tumor cells, although its cytotoxicity was slightly less than wt. In contrast, mut1 was shown to be much weaker in inducing VLS in mice than wt. The LD₅₀ values of wt and mut1 in mice were investigated with the result that the LD₅₀ of mut1 was about tenfold higher than that of wt. The in vivo antitumor activity of wt and mut1 were also compared in tumor-bearing nude mice. Both wt and mut1 were effective in inhibiting the tumor growth but mut1 showed improved therapeutic efficacy. These studies suggest mut1 may be a novel PE-based immunotoxin with much less toxicity for clinical use. Hao Wang, Shuichuan Song and Geng Kou contributed equally to this paper.     

Characterization of T-cell repertoire in hairy cell leukemia patients before and after recombinant immunotoxin BL22 therapy

We previously reported that hairy cell leukemia (HCL) patients have high percentages of CD56+/CD57+/CD3+ large granular lymphocytes consistent with cytotoxic T-lymphocytes (CTLs), and other investigators have reported skewing of the T-cell repertoire. In previous studies of up to seven HCL patients, many of the 22 established T-cell receptor (TCR) beta variable region (TRBV) families showed mono- or oligoclonal restriction. To determine whether percentages of CTLs are correlated with TRBV clonal excess, we studied 20 HCL patients with flow cytometry, PCR of TCR gamma and TRBV regions, and fractional gel electrophoresis of PCR-amplified TRBV CDR3 domains (CDR3 spectratyping). Increased percentages of CD3+/CD8+/CD57+ CTLs correlated with more mono/oligoclonal and fewer polyclonal TRBV families (r=0.53; P=0.016). Age correlated with number of mono/oligoclonal TRBV families (r=0.51; P=0.022). Time since last purine analog therapy correlated with number of polyclonal TRBV families (r=0.46; P=0.040), but treatment with the anti-CD22 recombinant immunotoxin BL22 was not related to clonal excess. We conclude that abnormalities in the T-cell repertoire in HCL patients may represent deficient immunity, and may be exacerbated by purine analogs. Increased CD3+/CD57+ T-cells may be a useful marker of abnormal TRBV repertoire in HCL patients, and might prove useful in deciding whether patients should receive biologic antibody-based treatment rather than repeated courses of purine analog for relapsed disease.     

In vitro evaluation ofPyrularia thionin—anti-CD5 immunotoxin

The membrane-active peptide,Pyrularia thionin, purified fromPyrularia pubera, was covalently conjugated to an anti-CD5 monoclonal antibody. The membrane-active properties of thionin were not affected by the conjugation. The immunotoxin killed CD5⁺ lymphocytes in vitro at a concentration of 0.1 nmol/10⁷ cells after 2 h of incubation. The immunotoxin also inhibited the proliferation of T cells in vitro, stimulated either by mitogens or in the mixed lymphocyte reaction. It was shown by electron paramagnetic resonance of spin probes and differential scanning calorimetry that the ability of the immunotoxin to perturb the lipid phase of membranes is close to that of unconjugated thionin. The results obtained suggest thatPyrularia-thionin—anti-CD5 conjugate may be useful for graft-versus-host disease therapy and potentially in the treatment of CD5⁺ leukemia and lymphomas.     

Immunotoxin studies in a model of human T-cell acute lymphoblastic leukemia developed in severe combined immune-deficient mice

The transplantation of the human T-cell acute lymphoblastic leukemia (T-ALL) cell line HSB-2 into severe combined immune-deficient (SCID) mice was found to produce a disseminated pattern of leukemia similar to that seen in humans. The iv injection of 10⁷ HSB-2 cells was associated with a universally fatal leukemia. Histopathological examination of animals revealed the spread of leukemia initially from bone marrow to involve all major organs including the meninges. An immunotoxin (HB2-Sap) was constructed by conjugating the anti-CD7 monoclonal antibody (MAb) HB2 to the ribosome inactivating protein (RIP) saporin. An in vitro protein synthesis inhibition assay revealed specific delivery of HB2-Sap immunotoxin (IT) to CD7⁺ HSB-2 target cells with an IC₅₀ of 4.5 pM. In an in vivo study, the IT was shown to significantly prolong the survival of SCID mice injected with HSB-2 cells compared to untreated control animals. This therapeutic effect was seen both with a single injection of 10 μg of IT given 7 d after the injection of HSB-2 cells, and was even more effective when IT was administered as three daily injections of 10 μg on d 7, 8, and 9. These results demonstrate the useful application of human leukemia xenografts in SCID mice and the potential therapeutic effect of an anti-CD7 IT in human T-ALL.     

Determination of human plasma xanthine oxidase activity by high-performance liquid chromatography

An assay for human plasma xanthine oxidase activity was developed with pterin as the substrate and the separation of product (isoxanthopterin) by high-performance liquid chromatography with a fluorescence detector. The reaction mixture consists of 60 μl of plasma and 240 μl of 0.2 M Tris-HCl buffer (pH 9.0) containing 113 μM pterin. With this assay, the activity of plasma xanthine oxidase could be easily determined despite its low activity. As a result, it could be demonstrated that the intravenous administration of heparin or the oral administration of ethanol did not increase plasma xanthine oxidase activity in normal subjects, and also that plasma xanthine oxidase activity was higher in patients with hepatitis C virus infection than in healthy subjects or patients with gout. In addition, a single patient with von Gierke's disease showed a marked increase in the plasma activity of this enzyme, relative to that apparent in normal subjects.     

Antitumour activity of a sterically blocked ricin immunotoxin on a human colorectal adenocarcinoma grafted subcutaneously in nude mice

Summary We prepared a ricin-antibody conjugate, lacking the ability to bind the galactosidic residues of Sepharose 6B, a so-called blocked immunotoxin. The monoclonal antibody AR-3 was cross-linked to ricin through a thioether bond. Further studies showed that the immunoconjugate suppressed the tumour growth of HT-29 cells in intraperitoneally grafted nude mice, without showing any undesirable ricin toxicity.In this work, to demonstrate the therapeutic activity of the AR-3—ricin conjugate injected into mice bearing subcutaneous tumour, we first evaluated its pharmacokinetic behaviour and biodistribution. The behaviour of the immunoconjugate injected intravenously was almost intermediate between that of the antibody and ricin. Moreover, when the immunotoxin was intravenously administered to nude mice bearing subcutaneous tumour, no therapeutic effects appeared, in accordance with the relatively low permeability of the immunotoxin from the blood to the skin. In contrast, peritumoral treatment produced a strong reduction of the neoplastic nodules without substantial regrowth of the malignant cells. This result was also achieved when the immunotoxin treatment was performed on a well-established tumour. This finding was strictly related to the specificity of the immunoconjugate, since the analogous treatment with an irrelevant immunotoxin showed therapeutic failure.     

Targeting malignant B cells with an immunotoxin against ROR1

The selective cell surface expression of receptor tyrosine kinase-like orphan receptor 1 (ROR1) in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) has made ROR1 a novel and promising target for therapeutic monoclonal antibodies (mAbs). Four mouse mAbs generated by hybridoma technology exhibited specific binding to human ROR1. Epitope mapping studies showed that two mAbs (2A2 and 2D11) recognized N-terminal epitopes in the extracellular region of ROR1 and the other two (1A1 and 1A7) recognized C-terminal epitopes. A ROR1- immunotoxin (BT-1) consisting of truncated Pseudomonas exotoxin A (PE38) and the V_H and V_L fragments of 2A2-IgG was made recombinantly. Both 2A2-IgG and BT-1 showed dose-dependent and selective binding to primary CLL and MCL cells and MCL cell lines. Kinetic analyses revealed 0.12-nM (2A2-IgG) to 65-nM (BT-1) avidity/affinity to hROR1, depicting bivalent and monovalent interactions, respectively. After binding to cell surface ROR1, 2A2-IgG and BT-1 were partially internalized by primary CLL cells and MCL cell lines, and BT-1 induced profound apoptosis of ROR1-expressing MCL cell lines in vitro (EC₅₀ = 16 pM–16 nM), but did not affect ROR1-negative cell lines. Our data suggest that ROR1-immunotoxins such as BT-1 could serve as targeted therapeutic agents for ROR1-expressing B cell malignancies and other cancers.     

Targeting malignant B cells with an immunotoxin against ROR1

The selective cell surface expression of receptor tyrosine kinase-like orphan receptor 1 (ROR1) in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) has made ROR1 a novel and promising target for therapeutic monoclonal antibodies (mAbs). Four mouse mAbs generated by hybridoma technology exhibited specific binding to human ROR1. Epitope mapping studies showed that two mAbs (2A2 and 2D11) recognized N-terminal epitopes in the extracellular region of ROR1 and the other two (1A1 and 1A7) recognized C-terminal epitopes. A ROR1- immunotoxin (BT-1) consisting of truncated Pseudomonas exotoxin A (PE38) and the V_H and V_L fragments of 2A2-IgG was made recombinantly. Both 2A2-IgG and BT-1 showed dose-dependent and selective binding to primary CLL and MCL cells and MCL cell lines. Kinetic analyses revealed 0.12-nM (2A2-IgG) to 65-nM (BT-1) avidity/affinity to hROR1, depicting bivalent and monovalent interactions, respectively. After binding to cell surface ROR1, 2A2-IgG and BT-1 were partially internalized by primary CLL cells and MCL cell lines, and BT-1 induced profound apoptosis of ROR1-expressing MCL cell lines in vitro (EC₅₀ = 16 pM–16 nM), but did not affect ROR1-negative cell lines. Our data suggest that ROR1-immunotoxins such as BT-1 could serve as targeted therapeutic agents for ROR1-expressing B cell malignancies and other cancers.     

Characterization of a recombinant monoclonal antibody by mass spectrometry combined with liquid chromatography

In this report, we present the characterization of a humanized monoclonal antibody specific for the human epidermal growth factor receptor (hEGFR). Direct analysis by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) of peptide mixtures and chromatographically isolated fractions allowed identification of 94.0% and 85.4% of the amino acid sequence of light and heavy chains, respectively. Microheterogeneity sources were identified in light and heavy chains and a previously unreported posttranslational modification for immunoglobulins was found. One N-glycosylation site was identified in the heavy chain with non-sialylated bianntenary fucosylated structures. This study is one of the first to assess the potential of MALDI-MS in combination with more conventional protein chemistry techniques for the characterization of monoclonal antibodies.     

Mechanism of cytolytic T lymphocyte killing of a low class I-expressing tumor

Many tumors have been shown to express minimal levels of class I MHC Ag, which makes them more resistant to recognition and lysis by cytolytic T lymphocytes. Line 1, a BALB/c spontaneous lung carcinoma, normally expresses very low levels of class I Ag, but expression can be increased 50-fold by treatment with agents such as DMSO or IFN-gamma. Because class I Ag serve as restricting elements for cytolytic T cell recognition of tumor Ag, we wished to determine if cytotoxic T lymphocytes could play a role in the immune response to this type of class I low, but inducible, tumor. After immunization in vivo and restimulation of splenic cells in vitro we were able to generate T cell clones that lysed line 1 cells induced to express high levels of class I, but did not lyse uninduced, low class I expressing line 1 cells in short term (6-h) 51Cr release assays. Paradoxically, incubation of the T cells with uninduced class I low line 1 cells for a few days resulted in complete destruction of the tumor cells. We demonstrate that the T cells, stimulated by the tumor cells, produce IFN-gamma, which in turn induces class I expression on the line 1 cells making them susceptible to lysis by the T cell clone. This suggests that a positive feedback reaction can occur in generating a response to this and perhaps other inducible tumor cell lines.     

A peptide mimetic of an anti-CD4 monoclonal antibody by rational design

The development of rational methods to design 'continuous' sequence mimetics of discontinuous regions of protein sequence has, to now, been only marginally successful. This has been largely due to the difficulty of constraining the recognition elements of a mimetic structure to the relative conformational and spatial orientations present in the parent molecule. Using peptide mapping to determine 'active' antigen recognition residues, molecular modeling, and a molecular dynamics trajectory analysis, we have developed a peptide mimic of an anti-CD4 antibody, containing antigen contact residues from multiple CDRs. The design described is a 27-residue peptide formed by juxtaposition of residues from 5 CDR regions. It displays an affinity for the antigen (CD4) of 0.9nM, compared to 2nM for the parent antibody ST40. Nevertheless, the mimetic shows low biological activity in an anti-retroviral assay.     

Engineering T lymphocyte antigen specificity.

Targeting of immune cells by bispecific antibodies has proven to be a powerful tool for the investigation of cellular cytotoxicity, lymphocyte activation and induction of cytokine production, as well as to represent an innovative form of immunotherapy for the treatment of cancer. The hallmark of this approach is the use of the specificity of monoclonal antibodies to join target and immune cells by virtue of the dual specificity of bispecific antibodies for the two entities. More precisely, the bispecific antibody has two different binding sites, which are capable of recognizing tumor associated antigens on the one hand and lymphocyte activation sites on the other. This process of crosslinking results in the activation of the lymphocyte and triggering of its lytic machinery, as well as lymphokine production. A major advantage of this therapeutic modality is, that use is made of the normal cellular immune defence system and therefore is only associated with minor toxicity. The distinct lymphocyte populations, which can be used for adoptive immunotherapy and the various bispecific antibody preparations, as well as the chimeric immunoglobulin/T cell receptor construction, are the major topics of this review.     

Expression, purification, and refolding of a novel immunotoxin containing humanized single-chain fragment variable antibody against CTLA4 and the N-terminal fragment of human perforin

Immunotoxins might be potential in treatment of cancer for their ability to kill selected cell populations. We constructed a novel immunotoxin hS83P34 by fusing N-terminal 34 amino acid fragment of human perforin to the C-terminus of humanized single-chain fragment variable antibody against CTLA4. The fusion protein was inductively expressed as inclusion bodies at a high level about 30% of total bacterial proteins. After washing with buffer containing 2 M urea, the purity of inclusion body was about 71%. The washed inclusion bodies were solubilized in 8 M urea and further purified to homogeneity (approximately 92% purity) by cation-exchange chromatography and Ni-agarose affinity chromatography under denaturing condition. The inclusion body refolding conditions were optimized following Pro-Matrix Protein Refolding Guide. After refolded in Tris buffer (pH 8.0) containing 1M urea, 0.8 M l-arginine, and 2 mM GSH:0.2 mM GSSG or 2 mM GSH:0.4 mM GSSG for 18h at 4 degrees C, over 90% proteins were recovered from inclusion bodies. In vitro dose-dependent cytotoxicity assay demonstrates that hS83P34 is only toxic to CTLA4-positive cells. IC(50) of hS83P34 for leukemic cells Raji and 6T-CEM are about 0.85 and 1.3 microM individually. Whereas, CTLA4-negative endothelial cell ECV-304 is resistant to hS83P34.     

Molecular cloning and characterisation of a novel xanthine oxidase from Cellulosimicrobium cellulans ATCC21606

Xanthine oxidase (XOD) catalyses the oxidation of hypoxanthine into xanthine and xanthine into uric acid. The enzyme plays a key role in the purine metabolic pathway. Despite the presence of different XODs in prokaryotes, the functional and structural knowledge of prokaryotic XODs remain limited (compared with their well-known eukaryotic counterparts), thereby hindering their biochemical analysis and industrial application. Using genetic and biochemical analyses, we identified and characterised recombinant XOD (CcXOD_AB) from Cellulosimicrobium cellulans ATCC21606. Bioinformatics analysis suggests that CcXOD_AB shares low amino acid sequence identities with other XODs. The purified enzyme exhibits the maximum activity at 55 °C and pH 8.0. In addition, CcXOD_AB exhibits moderate thermostability and retains 80.65 % of the original activity after 30 min of incubation at 60 °C. Ca^{2 +} has a slight inhibitory effect, whereas Co^{2 +} and Mn^{2 +} have a strong inhibitory effect on XOD_AB activity. In particular, low Ba²⁺ and Mg^{2 +} concentrations have no effect, whereas high Mg^{2 +} (≥10 mM) and Ba²⁺ (≥2 mM) concentrations show an inhibitory effect on enzyme activity. The K_m and V_max values for xanthine are 131.29 ± 11.09 μmol•L⁻¹ and 15.23 ± 0.65 μmol•L^-1 min⁻¹, respectively. Results indicate that CcXOD_AB is a novel enzyme with potential industrial application.     

应用CE-SDS测定抗CD52人源化单克隆抗体参比品单体纯度及非糖化重链比例

目的:测定抗CD52人源单克隆抗体参比品单体纯度及非糖基化重链比例。方法:采用PA800 plus毛细管电泳系统,非还原十二烷基苯硫酸钠-毛细管电泳(CE-SDS)测定抗CD52人源单克隆抗体单体纯度,以及用还原CE-SDS电泳测定非糖化重链比例。结果:非还原CE-SDS三次测定单体纯度平均值为93.57%,主峰的迁移时间及修正峰面积百分比RSD分别为0.16%和0.19%;还原CE-SDS电泳重链修正峰面积百分比平均值为66.89%,修正峰面积百分比及迁移时间RSD分别为0.09%和0%;轻链修正峰面积百分比平均值为32.30%;轻链修正峰面积及迁移时间RSD分别为0%和0%;非糖基化重链修正峰面积百分比平均值为0.87%,修正峰面积百分比及迁移时间RSD分别为6.66%和0.27%。结论:CE-SDS测定抗CD52人源单抗单体纯度及非糖化重链比例实验结果偏差较小,表明结果准确可靠。     

Production and introduction of a novel immunotoxin based on engineered RNase A for inducing death to Her1-positive cell lines

The present study was performed to design an immunotoxin consisting of engineered RNase A and scFv of Cetuximab. To accomplish this study goal, at first to evade RNase A from its inhibitors in the cytoplasm, six amino acids of RNase A were substituted, then the physicochemical features of engineered RNase A were assessed. To investigate the interaction between the engineered RNase A and the ribonuclease inhibitor, protein–protein docking was performed. After engineering the RNase A, it was theoretically conjugated with scFv of Cetuximab using a cleavable linker to produce scFv-engineered RNase A. Then, wild-RNase A (14 kD), engineered RNase A (14 kD) and scFv-engineered RNase A (42 kDa) were expressed in the BL21 (DE3) strain of Escherichia coli and purified by Ni-NTA columns. To confirm the expressed proteins, western blot analysis was performed. The functioning of wild-RNase A and engineered RNase A were investigated by RNA fragmentation assay. Finally, to evaluate the cytotoxicity of scFv-engineered RNase A, a dose–response cytotoxicity assay was performed on Her1-positive and Her1-negative cell lines. The results showed that engineered RNase A could maintain its structure and disulfide bonds and evade its inhibitor. Expression and purification were successfully conducted and both enzymes could degrade yeast RNA. The result of cytotoxicity showed that the engineered immunotoxin could induce cell death to Her1-positive cell lines with an IC₅₀ of 50 nM. It appears that scFv-engineered RNase A can be a promising molecule for use.     

Construction, expression, purification and functional analysis of recombinant 6C6 immunotoxin to human breast-tumor cells

The 28 ku membrane protein is usually over-expressl m human bnasl bmast cancer and other tumor cells. licould be a larget for tumor therapy . By using genetie engineermg teehmgues.a 606 immunotoxin (sefv606-PE40) was construeted by joining the 606 single-chain antibdy (SeFv606) with the truncll Pseudonwnas exotoxin A (PE40), SeFv606 contains both the heavy and light-chnia variable domams of 606 monoelonal antibody. Which speeifieally ree-ognizes the 28 ku protein. The bacterial expression level og 606 imnmmotoxin is 3.3%. about 5.5 mg ml baeterial lysate.lsing singlc-step llisTrap (Nr2 chelating) column chronnetogaphy, the reeombinant peptide was obtained with a purit of 33.2%.This baeterial espressed 606 immunotosin binuls to MDA-231 human breast-tumer ccll surfaee and kill these cells with a median lethal dosage of 92 ngnd.     

Posttranslational ruling of xanthine oxidase activity in bovine milk by its substrates

The aims of this study were to test the hypothesis that the substrates of xanthine oxidase (XO), xanthine and hypoxanthine, are consumed while the milk is stored in the gland between milkings, and to explore how XO activity responds to bacteria commonly associated with subclinical infections in the mammary gland. Freshly secreted milk was obtained following complete evacuation of the gland and induction of milk ejection with oxytocin. In bacteria-free fresh milk xanthine and hypoxanthine were converted to uric acid within 30 min (T1/2 approximately 10 min), which in turn provides electrons for formation of hydrogen peroxide and endows the alveolar lumen with passive protection against invading bacteria. On the other hand, the longer residence time of milk in the cistern compartment was not associated with oxidative stress as a result of XO idleness caused by exhaustion of its physiological fuels. The specific response of XO to bacteria species and the resulting bacteria-dependent nitrosative stress further demonstrates that it is part of the gland immune system.     

Development of a GMP Phase III purification process for VB4-845, an immunotoxin expressed in E. coli using high cell density fermentation

VB4-845 is a recombinant immunotoxin comprised of an anti-epithelial cell adhesion molecule (EpCAM) scFv fused to a truncated form of the bacterial toxin, Pseudomonas exotoxin A. VB4-845, purified from TB fed-batch fermentation, showed clinical efficacy when administered locally to treat non-muscle invasive bladder cancer (NMIBC) and squamous cell carcinomas of the head and neck (SCCHN). Here, we describe the implementation of an Escherichia coli high cell density (HCD) cultivation and purification process for VB4-845. HCD cultivation was a prerequisite for achieving higher yields necessary for Phase III clinical trials and commercialization. Using this process, the VB4-845 titer in the supernatant was increased by 30-fold over the original TB fed-batch cultivation. To obtain clinical grade material, a process involving a five-step column purification procedure was implemented and led to an overall recovery of ～ 40%. VB4-845 purity of >97% was achieved after the first three columns following the removal of low-molecular weight product-related impurities and aggregates. Endotoxins were effectively separated from VB4-845 on the Q-columns and by washing the Ni-column with a detergent buffer while host cell proteins were removed using ceramic hydroxyapatite. Comparability studies demonstrated that the purified product from the Phase III process was identical to the Phase II reference standard produced using TB fed-batch fermentation.     

Accessibility of an epitope common to all histone H3 variants in folded and unfolded chromatin as studied by a monoclonal antibody

In a recent publication the isolation and some characteristics of an anti-histone 3 monoclonal antibody, 1GB3 were described (Muller et al. FEBS Lett. 182: 459–464, 1985). We now report that the epitope recognized is phylogenetically conserved and located in the N-terminal part of H3, most likely between residues 40 and 50. Using the ELISA technique we found this region to be accessible in chromatin to the monoclonal antibody. The effect of non-ionic detergents on the adsorbtion of chromatin on microtiter plates was studied in this context.Immunological analysis of the reaction of the monoclonal antibody with chromatin by immunoinhibition and immunosedimentation shows that the H3 epitope is accessible in both folded and unfolded chromatin fibre as well as in high- and low-molecular weight oligonucleosomes.Abbreviations BSA Bovine srum albumin - mab Monoclonal antibody - PBS Phosphate buffered saline - PMSF Phenylmethyl sulfonyl fluoride