Pharmacokinetics of human-mouse chimeric anti-GD2 mAb ch14.18 in a phase I trial in neuroblastoma patients

A comprehensive analysis of the pharmacokinetics of human-mouse chimeric anti-ganglioside GD2 antibody mAb ch 14.18 was performed during a phase I clinical trial of ten children with neuroblastoma and one adult with osteosarcoma. The patients received a total of 20 courses of ch 14.18 at dose levels from 10 mg/m² to 200 mg/m². The plasma clearance of ch 14.18 was biphasic. Following the first course of treatmentt1/2, was 3.4±3.1 h andt1/2, 66.6±27.4 h in 9/10 children. Thet1/2, values were significantly less than those of 181±73 h previously reported in adult melanoma patients (P<-0.001), and 147.5 h in the adult osteosarcoma patient in our trial. The latter suggests different pharmacokinetics of mAb ch 14.18 in children and adults. After a second course of treatment, administered to 5/10 children,t1/2, decreased significantly from 72.9±19.8 h to 31.7±18.4 h (P=0.015). We there-fore conclude that the elimination kinetics of mAbs ch 14.18 in children and adults are different, and furthermore that repeated administration of mAb ch 14.18 to children with neuroblastoma leads to accelerated antibody clearance.This work was supported by grants from FDA, FD-R-000377 and NIH U10 CA 28439, and in part by a grant from the General Clinical Research Center Program, MOI RR00827, of the National Center for Research Resources, National Institutes of Health. M.M.U.-F. and C.S.H. were in part supported by a grant from the Children's Cancer Research Foundation, and M.M.U.-F. was in part supported by a grant of the Kommission für Forschung und wissenschaftlichen Nachwuchs, Charité 95-010/9610766     

Preliminary analgesic properties of deltorphin-5-methoxytryptamine chimeric opioid peptides

To further understand the relationship between melatonin (MT) and deltorphins (Dels) in pain modulation, two chimeric peptides (Del I-5-methoxytryptamine and Del II-5-methoxytryptamine) both containing 5-methoxytryptamine at the carboxyl-terminal of Dels mimicking MT were designed, synthesized and characterized by tail-flick assay in mice. Results showed that intracerebroventricular (i.c.v.) administration of Del I-5-methoxytryptamine (YaFDVVG-X, X is 5-methoxytryptamine, 5, 50 nmol/kg) or Del II-5-methoxytryptamine (YaFEVVG-X, X is 5-methoxytryptamine, 5, 50 nmol/kg) produced stronger analgesia than deltorphins (Del I or Del II alone), and acting even longer and stronger than cocktails containing Del I or Del II (50 nmol/kg) and MT (50 nmol/kg). Naloxone (i.p., 100 nmol/kg) could totally block the analgesic effects induced by the chimeric peptides, while luzindole (specific antagonist of melatonin receptor, i.p., 250 nmol/kg) could only partially inhibit the effects down to that induced by Dels alone. Interestingly, Del I-5-methoxytryptamine and Del II-5-methoxytryptamine act weaker with δ receptor than Dels in vitro but could induce much longer analgesia through co-activating δ opioid receptor and melatonin receptor.     

Construction of a chimeric aminopeptidase by a combination of gene shuffling and mutagenesis

Three chimeric genes were constructed by gene shuffling of aminopeptidases from Aeromonas caviae and Vibrio proteolyticus. Although expressed chimeric enzymes formed inclusion bodies in Escherichia coli, the introduction of two amino acid mutations into the chimeric genes by site-saturated mutagenesis and a random mutation on error-prone PCR resulted in solubilization of the chimeric enzyme. In addition, active chimeric enzyme showed a different thermostability and thermoactivity to parental enzymes.     

抗胃癌单抗3H11可变区氨基端序列对抗体活性的影响

采用RT-PCR方法,利用第一骨架区通用引物扩增重链Fd段和κ链的基因,克隆到Fab表达载体中,在大肠杆菌中获得了表达但未检测到抗原结合活性.根据已克隆的3H11 VL、VH的真实序列,重新设计κ链及Fd段5′端引物,分别将骨架区引物在κ链及Fd段5′端所造成的氨基酸残基改变纠正为原始序列,构建分别含有矫正后κ链或矫正后Fd以及二个链均得到矫正的Fab表达载体,这些载体在大肠杆菌中均获得类似水平的表达,对任何一个链的矫正均可部分恢复Fab段胃癌细胞的结合活性.结果说明在构建小分子抗体时,PCR引物引入的轻、重链可变区氨基端氨基酸残基的改变可严重影响所表达抗体的抗原结合活性.     

Digital twin of a continuous chromatography process for mAb purification: Design and model-based control

Model-based design of integrated continuous train coupled with online process analytical technology (PAT) tool can be a potent facilitator for monitoring and control of Critical Quality Attributes (CQAs) in real time. Charge variants are product related variants and are often regarded as CQAs as they may impact potency and efficacy of drug. Robust pooling decision is required for achieving uniform charge variant composition for mAbs as baseline separation between closely related variants is rarely achieved in process scale chromatography. In this study, we propose a digital twin of a continuous chromatography process, integrated with an online HPLC-PAT tool for delivering real time pooling decisions to achieve uniform charge variant composition. The integrated downstream process comprised continuous multicolumn capture protein A chromatography, viral inactivation in coiled flow inverter reactor (CFIR), and multicolumn CEX polishing step. An online HPLC was connected to the harvest tank before protein A chromatography. Both empirical and mechanistic modeling have been considered. The model states were updated in real time using online HPLC charge variant data for prediction of the initial and final cut point for CEX eluate, according to which the process chromatography was directed to switch from collection to waste to achieve the desired charge variant composition in the CEX pool. Two case studies were carried out to demonstrate this control strategy. In the first case study, the continuous train was run for initially 14 h for harvest of fixed charge variant composition as feed. In the second case study, charge variant composition was dynamically changed by introducing forced perturbation to mimic the deviations that may be encountered during perfusion cell culture. The control strategy was successfully implemented for more than ±5% variability in the acidic variants of the feed with its composition in the range of acidic (13%–17%), main (18%–23%), and basic (59%–68%) variants. Both the case studies yielded CEX pool of uniform distribution of acidic, main and basic profiles in the range of 15 ± 0.8, 31 ± 0.3, and 53 ± 0.5%, respectively, in the case of empirical modeling and 15 ± 0.5, 31 ± 0.3, and 53 ± 0.3%, respectively, in the case of mechanistic modeling. In both cases, process yield for main species was >85% and the use of online HPLC early in the purification train helped in making quicker decision for pooling of CEX eluate. The results thus successfully demonstrate the technical feasibility of creating digital twins of bioprocess operations and their utility for process control.     

Synthesis and biological properties of chimeric interferon-alpha2b peptides

We have previously reported the antiproliferative activity of synthetic sequences 29-35 and 122-139 of the interferon-alpha2b (IFN-alpha2b), both probably representing a common receptor recognition domain. In the search of new peptidic agonists, we designed and synthesized the linear peptide (Gly)2-122-137-Gly138-Gly29-30-35-(Gly)2, in which Gly residues replaced the 138 and 29 Cys bound through a disulfide bridge in the native cytokine. Additionally, a cyclic analog was obtained by reaction of the N- and C-terminal ends of the linear fragment. Thus, the distance that separates residues 122 and 35 in the crystalline structure of the IFN-alpha2b was maintained through a (Gly)4 bridge. When the influence of chimeric peptides on the proliferation of WISH cells was studied, it was shown that both derivatives significantly diminished cell growth. A more evident inhibitory effect on (125)I-IFN-alpha2b binding to WISH cell-membrane receptors was observed for both peptides. Results indicated that chimeric IFN-alpha2b peptides behaved as partial agonists of the IFN-alpha2b molecule and may be of interest for drug design purposes.     

Characterization of a novel modification of a CHO-produced mAb: Evidence for the presence of tyrosine sulfation

Herein we describe the investigation of a Chinese hamster ovary (CHO)-expressed human mAb molecule found partially modified by a +80 Da adduct. This mass difference, suggestive of a single sulfation or phosphorylation addition, was observed by mass analysis of the intact and reduced molecule by mass spectrometry (MS). The modification was located on tyrosine 31 (Y31) of the light chain in the complementarity-determining region 1 by liquid chromatography (LC)-MS peptide mapping and electron transfer dissociation fragmentation. The complete loss of the 80 Da modification moiety during collision induced dissociation fragmentation suggested this modification could not be a tyrosine phosphorylation. Treatment of the mAb with alkaline phosphatase confirmed our hypothesis. Western blot experiment using anti-tyrosine sulfation antibody and LC retention time correlation with corresponding synthetic sulfated peptides further confirmed the identification of tyrosine sulfation on the light chain. The unique sequence motif with neighboring acidic amino acids and local secondary structure might play a role to make Y31 a substrate residue for sulfation. This type of modification, to our knowledge, has not been previously reported for CHO-produced human IgG antibodies.     

Antitumour activity of a chimeric antibody against the leucocyte antigen CD48

Preclinical studies with the murine anti-CD48 antibody, mHuLym3 (IgG2a) have shown it to be a potentially useful therapeutic reagent in the treatment of human leukaemia and lymphoma. For clinical use, humanised antibodies can have a number of advantages over their original murine version, including mediation of higher effector cell function with human cells, longer serum half-life and lower immunogenicity. In this study, we have produced a mouse/human chimeric HuLym3 antibody (cHuLym3) where the murine antibody constant regions have been replaced with human constant regions. We report the production and preclinical characterisation of the antibody, cHuLym3, with potent in vitro and in vivo antitumour activity. The genes encoding the variable heavy and light chains were amplified by the polymerase chain reaction, sequenced and cloned into eukaryotic expression vectors containing the human light- and heavy-chain constant regions (κ and IgG1). The chimeric and murine HuLym3 antibodies had similar cell-binding specificity and affinity. In the human Raji cell severe combined immunodeficient mouse model the i.v. injection of cHuLym3 and mHuLym3 produced similar antitumour responses. Doses of cHuLym3 and mHuLym3 (100 μg) on days 1, 2 and 4 after i.v. Raji cell injection produced a 40% longer time to hind-leg paralysis than when a control antibody was used. cHuLym3 had more potent activity than mHuLym3 in antibody-dependent cellular cytotoxicity (ADCC) assays in vitro, with human peripheral blood mononuclear cells as effectors. Up to 60% specific cell lysis was observed with cHuLym3 in ADCC assays. These properties suggest that anti-CD48 antibodies may be useful in the treatment of a number of diseases including lymphoid leukaemias and lymphoma. Received: 5 May 1999 / Accepted: 12 August 1999     

Structural analysis of chick ephrin-A2 by function-blocking and non-blocking monoclonal antibodies

Ephrins, ligands for the Eph family of receptor tyrosine kinases, play key roles in diverse biological processes. In this study, we determined the epitopes and kinetic parameters of function-blocking (B3) and non-blocking (IV) monoclonal antibodies (mAbs) recognizing chick ephrin-A2. We show that the epitope for the non-blocking mAb is the residue Asp(105) of chick ephrin-A2. However, the binding of the function-blocking mAb depends mostly on residue Ser(108) and its epitope may reside within residues 105-132, which appear crucial for the receptor interaction site. Kinetic studies suggest a possible mechanism why mAb IV, despite recognizing a region very close to the mAb B3 epitope, fails to block the ligand-receptor interaction.     

Characterization of epitope regions of thyrotropin beta-subunit recognized by the monoclonal antibodies mAb279 and mAb299: a chimeric peptide approach.

This investigation describes the design, synthesis and evaluation of chimeric peptides related to the bovine thyrotropin beta-subunit, bTSHbeta. The structures of these chimeric peptides were derived from investigations with linear peptides and sequence alignment studies, in association with a homology model of TSHbeta developed from the hCG X-ray crystallographic structure. The structures of these chimeric peptides comprised beta-turn regions of loop L1 [bTSHbeta(14-20)] and loop L3 [bTSHbeta(65-72)] held in close proximity by a bis-beta-alanine linker and the disulfide bond bTSHbeta[Cys16-Cys67]. Linear and cyclic chimeric peptides were evaluated in immunochemical assays for their ability to inhibit the binding of radio-iodinated bTSHbeta [125I-bTSHbeta] to the monoclonal antibodies, mAb279 and mAb299. Previously, mAb279 and mAb299 have been shown to recognize epitopes accessible on the surface of TSHbeta that lie in close proximity to the TSH receptor-binding site. The results indicate that these chimeric peptides can specifically inhibit in a dose-dependent manner the binding of 125I-bTSHbeta to mAb299, while having a lesser effect on the binding with mAb279. Based on these results, it can be concluded that the bTSHbeta-epitope recognized by mAb299 involves contributions from amino residues from the beta-turn regions of the L1 and L3 loops of TSHbeta, and that these loop regions flank part of the receptor binding site of the bTSH beta-subunit.     

Construction and functional characteristics of tuber-specific and cold-inducible chimeric promoters in potato

The improvement of processing quality of potato products (fries and chips) demands less accumulation of reducing sugars (glucose and fructose) in cold-stored potato (Solanum tuberosum) tubers. Control of gene expression to achieve this requires promoters with specificity to tubers as well as inducible activity under low temperatures. Here we use overlapping extension PCR to construct two chimeric promoters, pCL and pLC, to control gene expression in a tuber-specific and cold-inducible pattern. This combined different combinations of the LTRE (low-temperature responsive element) from Arabidopsis thaliana cor15a promoter and the TSSR (tuber-specific and sucrose-responsive sequence) from potato class I patatin promoter. The cold-inducible and tuber-specific activities of the chimeric promoters were investigated by quantitative analysis of GUS activity in transgenic potato cultivar E3 plants. The results showed that the cis-elements, LTRE and TSSR, played responsive roles individually or in combination. pCL with the TSSR closer to the TATA-box showed substantially higher promoter activity than pLC with the LTRE closer to the TATA-box at either normal (20°C) or low temperature (2°C), suggesting that the promoter activity was closely associated with the position of the two elements. The chimeric promoter pCL with tuber-specific and cold-inducible features may provide valuable tool for controlling the expression of gene constructs designed to lower the formation of reducing sugars in tubers stored at low temperature and to improve the processing quality of potato products. The nucleotide sequence data reported will appear in the GenBank database under the accession numbers DQ494557 (pCL) and DQ494558 (pLC ).     

HCV的Core-NS3嵌合抗原在大肠杆菌中的表达、纯化及初步应用

用已经构建的含有HCV的Core-NS3(C33 c)嵌合基因的表达质粒pGEX TL1-2在大肠杆菌中进行了高效表达,表达产物纯化后通过SDS-PAGE、W estern-b lot、ELISA等一系列鉴定试验进行了分析,结果表明,该嵌合抗原具有高度特异性和良好的抗原活性,为研制诊断用HCV抗原奠定基础。     

Anti-Pim-1 mAb inhibits activation and proliferation of T lymphocytes and prolongs mouse skin allograft survival

Pim-1 is an important signaling molecule mediating cell proliferation and survival. Our previous study identified a Pim-1 specific monoclonal antibody, P9, with significant inhibitory effect on cell proliferation. Herein, we report that P9 inhibited the activation and proliferation of PHA-stimulated human PBMC and induced them to undergo apoptosis. In contrast, P9 showed little effect on freshly isolated human blood T lymphocytes which poorly expressed Pim-1. P9 also detected an up-regulation of Pim-1 in mouse lymphocytes after mitogen stimulation, and showed similar selective inhibition on stimulated cells as observed with hPBMC. Furthermore, P9 inhibited the in vitro mixed lymphocyte reaction and P9 treatment significantly prolonged the survival of mouse skin allografts (P < 0.001). It is concluded that Pim-1 expression correlates with lymphocyte proliferation and activation. P9 functions as a Pim-1 antagonist and is potential for immunosuppressive therapy.     

Creation and analysis of a novel chimeric promoter for the complete containment of pollen- and seed-mediated gene flow

Effective containment of gene flow in transgenic plants requires a promoter that is highly specific for male and female gametes or tissues. Here, we report the creation of a novel pollen-, stigma- and carpel-specific (PSC) promoter through the fusion of the pollen-specific LAT52 and carpel-specific AGL5 enhancers to a stigma-specific SLG promoter. Gene expression analysis showed that fusion of the LAT52 enhancer to the SLG promoter enables the latter to gain pollen-specific activity while the acquirement of carpel-specific activity requires the correct orientation of the inserted AGL5 enhancer in the PSC promoter, and only a forward- but not a reverse-oriented one is functional. The resulting fPSC promoter, when fused to DT-A, generated at least three aberrant gynoecium phenotypes. Type I plants exhibited shortened stigmatic tissues, resembling plants containing the DT-A gene controlled by the SLG promoter. However, type II and III plants displayed partial or complete ablation of gynoecia, and were unable to support the reproductive process. Type II and III plants also produced severely perturbed anthers and pollen in comparison to type I or SLG::DT-A plants, and transgenic pollen grains were unable, when out-crossed with control plants, to pass the transgene to the next generation in all plants examined, indicating that they are selectively eliminated. This tissue-specific ablation or perturbation is highly specific, and does not compromise vegetative growth. Evidently, the fPSC promoter faithfully acquires tissue specificity from the incorporated enhancers and promoter, and should have a practical application for transgene containment in non-fruit and -grain producing plant crops.     

Increased biological activity of a recombinant factor IX variant carrying alanine at position +1

In attempts to improve the post-translational modification and processing of recombinant factor IX (FIX) we have altered the cDNA sequence encoding pre-pro-FIX using site-directed mutagenesis and have expressed the variant cDNAs in BHK21 cells using a vaccinia-virus-derived vector. We find that substitution of the tyrosine residue at +1 for an alanine increases the biological activity of the recombinant molecules 2-fold. On the other hand, substitution of the proline at -3 for a valine results in no significant change to the specific activity of the protein. Other alterations to the N-terminus of the FIX proteins, in attempts to mimic other vitamin-K-dependent proteins, result in the failure to produce a secreted polypeptide. N-terminal sequence analysis of purified recombinant molecules reveals a correlation between specific activity and the efficiency of correct pro-sequence cleavage. gamma-Carboxylation analysis of purified recombinant proteins indicates that each molecule including unmutated FIX is completely gamma-carboxylated in this system. Thus the observed increase in biological activity of FIX variants containing an alanine at position +1 is not due to increased gamma-carboxylation but, at least in part, to more efficient pro-peptide cleavage.     

Monocot chimeric jacalins: a novel subfamily of plant lectins

Abstract

Monocot chimeric jacalins are a small group of lectins (currently with nine members), each typically consisting of a dirigent domain and a jacalin-related lectin domain. This unique module structure, along with their limited taxonomic distribution and short time window in molecular evolution, makes them a novel family of lectins. Recent studies have shown that these proteins play important roles in plant stress responses and development. Our knowledge of these proteins in functional domain and evolution has also made significant progress.     

Pharmacokinetics of anti-ganglioside GD2 mAb 14G2a in a phase I trial in pediatric cancer patients

A phase I trial of a murine anti-ganglioside (GD2) monoclonal antibody (mAb) 14G2a was conducted in 14 neuroblastoma patients and 1 osteosarcoma patient to assess its safety, toxicity and pharmacokinetics in pediatric patients. The pharmacokinetics of mAb 14G2a were biphasic with at ₁ ^2/ of 2.8±2.8 h and at ₁ ^2/ of 18.3±11.8 h. In general,t ₁ ^2/ was dose-dependent with a level of significance ofP=0.036, and it reached a plateau at doses of 250 mg/m² or more. Overall the peak serum levels were dose-dependent atP<0.001. However, they demonstrated an abrupt increase between doses of 100 mg/m² and 250 mg/m². The latter two suggest a saturable mechanism for mAb elimination. In addition, peak serum concentrations were observed earlier at higher mAb doses, which indicates the achievement of a steady state. Thet ₁ ^2/ of mAb 14G2a in children appears to be shorter than in adults. Furthermore, 2 patients demonstrated a considerable decrease int ₁ ^2/ following retreatment with 14G2a. This was paralleled by high human anti-(mouse Ig) antibody levels. This study represents the first comprehensive analysis of murine mAb pharmacokinetics in children and will be useful in the future design of mAb therapy.This work was supported by grants from FDA, FD-R-000377 and NIH U10 CA 28439 and in part by a grant from the general Clinical Research Center program, MOI RR00827, of the National Center for Research Resources, National Institutes of Health. M. M. U.-F. and C.-S. H. were supported in part by a grant from the Children's Cancer Research Foundation, and R. A. R. was supported in part by NIH grant CA 42508     

Mapping effector functions of a monoclonal antibody to GD3 by characterization of a mouse-human chimeric antibody

R24, a mouse monoclonal antibody against GD3 ganglioside, exhibits a wide range of in vitro effector functions. It also has the ability to bind to itself, presumably through homophilic Fab-Fab interactions, which have been proposed to contribute to its high relative avidity for GD3 and to its effector function activity. It is not known which of these characteristics is necessary for the antitumor effects observed in melanoma patients treated with R24. A mouse-human chimeric R24 (chR24) molecule has been constructed in which the GD3-binding site is preserved. Chimeric R24 demonstrates a lower level of binding to GD3 than does mouse R24 suggesting that there may be some differences between the GD3-binding sites of the two mAb or that Fc determinants can contribute to R24 avidity for GD3. The property of homophilic binding is retained by chR24, demonstrating formally that homophilic binding of R24 involves interactions between variable domains. Both R24 and chR24 fix human complement and mediate antibody-dependent cellular cytotoxicity although chR24 was slightly less efficient at the latter. Unlike R24, chR24 was not able to inhibit melanoma cell attachment to plastic surfaces and was not able to activate human T lymphocytes. We hypothesize that chR24 does not bind to GD3 with an avidity high enough to mediate these effector functions.Supported by Public Health Service grants PO1-CA33049 and RO1-CA57363     

In vitro and in vivo antitumour activity of a chimeric anti-CD19 antibody

Mouse monoclonal antibodies to CD19 detect an antigenic determinant expressed exclusively on the surface of B lymphocytes, and have previously been shown to be potentially useful therapeutic reagents for human B cell lymphoma. We report the production and characterization of a mouse/human chimeric antibody, cCD19, with potent in vivo antitumour activity. The genes encoding the variable domains for heavy (V_H) and light (V_L) chains were subcloned into eukaryotic expression vectors containing human constant region genes (IgG1 and ), and co-transfected into non-secreting Sp2/0 mouse myeloma cells. Intraperitoneal administration of cCD19 produced inhibition of growth of subcutaneous CD19⁺ Sultan human B lymphoma tumours inscid/scid mice. When the antibody was administered 18 and 20 days after subcutaneous tumour inoculation, an approximately 30% reduction in tumour size was noted by day 29. cCD19 faithfully mimicked the in vitro binding characteristics of mCD19 as (a) the chimeric antibody was shown by flow cytometry to bind exclusively to cell lines that expressed CD19, (b) cCD19 was able to inhibit the binding of mCD19 on CD19⁺ cells completely and (c) the affinity of binding of the two antibodies was not significantly different [K _a=(2.03±1.5)×10⁸]. In biodistribution studies, up to 14.8% of the total injected antibody dose per gram of tissue was localized in CD19⁺ Sultan tumours at 24 h approximately, 14.4% was present in the tumors at 48 h and about 13.7% at 72 h. These levels were comparable to mCD19 administered in the same fashion. cCD19 conjugated to idarubicin was specifically and strongly cytotoxic to CD19⁺ cells cultured in vitro, and demonstrated an IC₅₀ of 0.17 M, similar to that of mCD19 (0.32 M) and approximately 14-fold greater than the IC₅₀ of free idarubicin. The specific cytotoxic capacity of cCD19 and its likely reduced immunogenicity suggest that it may potentially be of use in the treatment of refractory B cell lymphoma in humans.