Targeting myeloid leukemia with a DT(390)-mIL-3 fusion immunotoxin: ex vivo and in vivo studies in mice.

The IL-3 receptor was expressed on a high frequency of myeloid leukemia cells and also on hematopoietic and vascular cells. We previously showed that a recombinant IL-3 fusion immunotoxin (DT(390)IL-3) expressed by splicing the murine IL-3 gene to a truncated diphtheria toxin (DT(390)) gene selectively killed IL-3R(+) expressing cells and was not uniformly toxic to uncommitted BM progenitor cells (Chan,C.-H., Blazar,B.R., Greenfield,L., Kreitman,R.J. and Vallera,D.A., 1996, Blood, 88, 1445-1456). Thus, we explored the feasibility of using DT(390)IL-3 as an anti-leukemia agent. DT(390)IL-3 was toxic when administered to mice at doses as low as 0.1 microg/day. The dose limiting toxicity appeared to be related to platelet and bleeding effects of the fusion toxin. Because of these effects, DT(390)IL-3 was studied ex vivo as a means of purging contaminating leukemia cells from BM grafts in a murine autologous BM transplantation. In this setting, as few as 1000 IL-3R-expressing, bcr/abl transformed myeloid 32Dp210 leukemia cells were lethal. An optimal purging interval of 10 nM/l for 8 h eliminated leukemia cells from 32Dp210/BM mixtures given to lethally irradiated (8 Gy) C3H/HeJ syngeneic mice. Mice given treated grafts containing BM and a lethal dose of 32Dp210 cells survived over 100 days while mice given untreated grafts did not survive (P < 0.00001). DT(390)IL-3 may prove highly useful for ex vivo purging of lethal malignant leukemia cells from autologous BM grafts.     

Development of a diphtheria toxin based antiporcine CD3 recombinant immunotoxin

Anti-CD3 immunotoxins, which induce profound but transient T-cell depletion in vivo by inhibiting eukaryotic protein synthesis in CD3+ cells, are effective reagents in large animal models of transplantation tolerance and autoimmune disease therapy. A diphtheria toxin based antiporcine CD3 recombinant immunotoxin was constructed by fusing the truncated diphtheria toxin DT390 with two identical tandem single chain variable fragments (scFv) derived from the antiporcine CD3 monoclonal antibody 898H2-6-15. The recombinant immunotoxin was expressed in a diphtheria-toxin resistant yeast Pichia pastoris strain under the control of the alcohol oxidase promoter. The secreted recombinant immunotoxin was purified sequentially with hydrophobic interaction chromatography (Butyl 650 M) followed by strong anion exchange (Poros 50 HQ). The purified antiporcine CD3 immunotoxin was tested in vivo in four animals; peripheral blood CD3+ T-cell numbers were reduced by 80% and lymph node T-cells decreased from 74% CD3+ cells pretreatment to 24% CD3+ cells remaining in the lymph node following 4 days of immunotoxin treatment. No clinical toxicity was observed in any of the experimental swine. We anticipate that this conjugate will provide an important tool for in vivo depletion of T-cells in swine transplantation models.     

Expression of an anti-CD3 single-chain immunotoxin with a truncated diphtheria toxin in a mutant CHO cell line

ADP-ribosylating immunotoxins are generally expressed in Escherichia coli and then refolded in vitro. Because the efficiency of the in vitro refolding process decreases with the number of protein domains and internal disulfide bonds, these immunotoxins have been generally limited to single-chain monovalent structures. We now show that using the hamster cell line CHO K1 RE1.22c (J. M. Moehring and T. J. Moehring, 1979, Somat. Cell Genet. 5, 453-468) that has been mutated to ADP-ribosylation insensitivity, a level of 4 microg/ml of a truncated anti-T cell immunotoxin, DT390-scFvUCHT1, can be secreted into the medium. This immunotoxin is glycosylated at the two potential N-linked glycosylation sites in the toxin moiety: positions 16-18 in the A chain and residues 235-237 in the B chain. The glycosylated immunotoxin is relatively nontoxic (IC(50) 4.8 x 10(-10) M). Removal of the N-linked oligosaccharides by N-glycosidase F treatment or mutations at the two N-linked glycosylation sites results in a highly active immunotoxin with an IC(50) of 4 x 10(-12) M toward CD3(+) Jurkat cells. This is a 12-fold increase in toxicity over the same immunotoxin harvested from E. coli periplasm without refolding. A single Asn(235) Ala mutation that removed the B chain glycosylation was nearly as toxic as the double mutant. This suggests that B chain glycosylation is the major cause for the loss of toxicity.     

Targeting glioblastoma multiforme with an IL-13/diphtheria toxin fusion protein in vitro and in vivo in nude mice

Fusion proteins composed of tumor binding agents and potent catalytic toxins show promise for intracranial therapy of brain cancer and an advantage over systemic therapy. Glioblastoma multiforme (GBM) is the most common form of brain cancer and overexpresses IL-13R. Thus, we developed an interleukin-13 receptor targeting fusion protein, DT(390)IL13, composed of human interleukin-13 and the first 389 amino acids of diphtheria toxin. To measure its ability to inhibit GBM, DT(390)IL13 was tested in vitro and found to inhibit selectively the U373 MG GBM cell line with an IC(50) around 12 pmol/l. Cytotoxicity was neutralized by anti-human-interleukin-13 antibody, but not by control antibodies. In vivo, small U373 MG glioblastoma xenografts in nude mice completely regressed in most animals after five intratumoral injections of 1 microg of DT(390)IL13 q.o.d., but not by the control fusion protein DT(390)IL-2. DT(390)IL13 was also tested against primary explant GBM cells of a patient's excised tumor and the IC(50) was similar to that measured for U373 MG. Further studies showed a therapeutic window for DT(390)IL13 of 1-30 microg/injection and histology studies and enzyme measurements showed that the maximum tolerated dose of DT(390)IL13 had little effect on kidney, liver, spleen, lung and heart in non-tumor-bearing immunocompetent mice. Together, these data suggest that DT(390)IL13 may provide an important, alternative therapy for brain cancer.     

重组人白细胞介素-3真核表达质粒直接注射小鼠骨骼肌的体内表达研究

将含有人白细胞介素-3基因(hIL-3)cD-NA的真核表达质粒直接注射小鼠骨骼肌,观察hIL-3基因在小鼠肌肉及体内的分泌表达情况。原位分子杂交及免疫组化结果显示注射重组质粒后,第14大重组质粒全部进入到骨骼肌细胞内并有IL-3的表达。ELISA结果显示,注射重组质粒DNA后,第21天小鼠血液中IL-3的含量最高,约有67ng/ml,第14天次之,约51ng/ml,第28天和第7天相近,约34ng/ml。生物学活性检测结果表明,实验组小鼠血清有维持IL-3依赖的TF-1细胞株生长的作用。表明重组质粒DNA直接注射骨骼肌后进入到肌细胞内并表达IL-3,进入血液,而且表达产物有生物学活性。     

IL—18DNA免疫对HIV—1核酸疫苗诱导的免疫应答的影响

为了研究白细胞介素-18(IL-18)基因对人免疫缺陷病毒(HIV-1)核酸疫苗诱导免疫应答的影响,将人IL-18基因插入到真核表达载体pVAX1中,构建了真核表达载体pVAX1-IL-18;将pCI-neoGAG联合pVAX1-IL-18或者pCI-neoGAG单独免疫Balb/c小鼠,检测免疫小鼠的特异性抗体和IFN-γ,同时观察免疫小鼠脾淋巴细胞增殖和小鼠特异性细胞毒性T淋巴细胞(CTL)反应。酶切及测序结果表明成功地构建了人IL-18基因真核表达载体;与pCI-neoGAG免疫组比较,pCI-neoGAG联合pVAX1-IL-18免疫组小鼠血清的抗HIV-1p24抗体滴度降低(P<0.01);而与pCI-neoGAG免疫组比较,pCI-neoGAG联合pVAX1-IL-18免疫组小鼠血清的IFN-γ升高(P<0.01);pCI-neoGAG联合pVAX1-IL-18免疫组小鼠的脾淋巴细胞增殖实验刺激指数(SI)以及特异性CTL活性均高于pCI-neoGAG免疫组(P<0.01)。IL-18基因联合HIV-1核酸疫苗免疫小鼠,可能增强特异性Th1细胞和CTL反应,白细胞介素-18基因对体液免疫有抑制作用。     

Characterization of a T suppressor cell line that downgrades experimental allergic encephalomyelitis in mice.

A T-suppressor (Ts) cell line of CD8 phenotype was isolated from spleens of SJL/J mice that had recovered from experimental allergic encephalomyelitis (EAE) induced by injection of MBP-activated T cells. The Ts cell line inhibited the proliferation of MBP-sensitized T cells in vitro. Addition of recombinant IL-2 enhanced the Ts-mediated suppression. Adoptively transferred Ts line was able to downgrade EAE in mice subsequently challenged with MBP-activated T cells. The mechanism of suppression appeared to involve neither direct cytolysis of the effector T cells nor the production of a soluble suppressor factor. The findings suggest an in vivo role for suppressor T cells in the regulation of EAE.     

Improved binding of a bivalent single-chain immunotoxin results in increased efficacy for in vivo T-cell depletion.

Anti-CD3 immunotoxins exhibit considerable promise for the induction of transplantation tolerance in pre-clinical large animal models. Recently an anti-human anti-CD3epsilon single-chain immunotoxin based on truncated diphtheria toxin has been described that can be expressed in CHO cells that have been mutated to diphtheria toxin resistance. After the two toxin glycosylation sites were removed, the bioactivity of the expressed immunotoxin was nearly equal to that of the chemically conjugated immunotoxin. This immunotoxin, A-dmDT390-sFv, contains diphtheria toxin to residue 390 at the N-terminus followed by VL and VH domains of antibody UCHT1 linked by a (G(4)S)(3) spacer (sFv). Surprisingly, we now report that this immunotoxin is severely compromised in its binding affinity toward CD3(+) cells as compared with the intact parental UCHT1 antibody, the UCHT1 Fab fragment or the engineered UCHT1 sFv domain alone. Binding was increased 7-fold by adding an additional identical sFv domain to the immunotoxin generating a divalent construct, A-dmDT390-bisFv (G(4)S). In vitro potency increased 10-fold over the chemically conjugated immunotoxin, UCHT1-CRM9 and the monovalent A-dmDT390-sFv. The in vivo potency of the genetically engineered immunotoxins was assayed in the transgenic heterozygote mouse, tgepsilon 600, in which the T-cells express human CD3epsilon as well as murine CD3epsilon. T-cell depletion in the spleen and lymph node observed with the divalent construct was increased 9- and 34-fold, respectively, compared with the monovalent construct. The additional sFv domain appears partially to compensate for steric hindrance of immunotoxin binding due to the large N-terminal toxin domain.     

鸭白介素-18基因重组真核表达载体的构建及其表达产物的生物学活性

从克隆质柱pGEM-DuIL-18扩增出鸭IL-18全基因片段,克隆到真核表达载体pcDNA3.1( )中,构建重组质粒pcDNA3.1/DuIL-18(简称pDuIL-18).将重组质粒pDuIL-18转染Cos7细胞,转染细胞中含鸭IL-18基因的mRNA.SDS-PAGE分析表明,表达产物是与鸭IL-18相符的约23 000的蛋白条带.鸭淋巴细胞转化试验表明,表达产物对鸭淋巴细胞具有明显诱导转化作用.重组质柱pDuIL-18对H<,9>亚型禽流感灭活疫苗免疫增强作用的研究表明重组质粒pDuIL-18能够提高禽流感灭活疫苗诱发的细胞免疫应答,为研究能够更好地防制禽流感的新型疫苗提供了新的思路.     

Cancer targeting gene-viro-therapy for pancreatic cancer using oncolytic adenovirus ZD55-IL-24 in immune-competent mice

Cancer targeting gene-viro-therapy (CTGVT) may prove to be an effective treatment for pancreatic cancer (PC). This study was intended to explore the anti-tumor effect of ZD55-IL-24 (oncolytic adenovirus ZD55 harboring IL-24) on PC in immune-competent mice. The expression of gene harbored by oncolytic adenovirus ZD55 in PC cells was detected by reporter-gene assays. The in vitro anti PC ability of ZD55-IL-24 was tested by MTT, crystal violet staining and apoptosis assays. The in vivo anti PC effect of ZD55-IL-24 was further observed in an immune-competent mice model by detecting anti-tumor immunity and induction of apoptosis. The expression of gene harbored by ZD55 in PC cells was significantly higher than that harbored by the replicated-deficient adenovirus, and the amount of gene expression was time-dependent and dose-dependent. Both ZD55-IL-24 and ZD55 inhibited PC cells growth, but the anti-tumor effect of ZD55-IL-24 was significantly stronger than that of ZD55, and the ability of ZD55-IL-24 in inducing PC apoptosis was significantly stronger than that of ZD55. The tumor-forming rate of group ZD55-IL-24 was the lowest, and the tumor-growing rate was also significantly lower than that of group ZD55 in immune-competent PC models. Moreover, ZD55-IL-24 mediated more anti-cancer immunity effects by induction of stronger T-lymphocytes response to PC cells, higher levels of γ-IFN and IL-6 cytokines. ZD55-IL-24-mediated CTGVT could inhibit PC growth not only by inducing oncolysis and apoptosis but enhancing the anti-cancer immune effects by inducing T cell response to PC and up-regulating γ-IFN and IL-6 cytokine in immune-competent mice. This may serve as a candidate therapeutic approach for the treatment of PC.     

Ad-ING4-IL-24 双基因共表达对人肺腺癌化疗的增敏效应

研究ING4 (肿瘤生长抑制因子4) 和IL-24 (人白细胞介素24) 双基因共表达腺病毒载体 (Ad-ING4-IL-24) 对肺腺癌的化疗增敏效应及分子机制。将Ad-ING4-IL-24感染A549肺癌细胞及联合顺铂 (DDP) 化疗药物作用,RT-PCR和Western blotting检测ING4和IL-24基因在A549肺癌细胞中的转录和表达,MTT法、流式细胞仪 (FCM) 和 Hoechst 染色法检测Ad-ING4-IL-24联合DDP (联合组) 对A549肺癌细胞的生长抑制和凋亡效应。采用A549细胞株建立人肺腺癌裸鼠模型,然后瘤体局部注射干预用药,动态测量肿瘤体积,并计算瘤重抑瘤率,免疫组化检测ING4、IL-24、bax、bcl-2、VEGF等基因的表达。结果表明,Ad-ING4-IL-24感染A549肺癌细胞后可获得成功转录和表达,体外联合组能明显抑制A549肺癌细胞生长和诱导细胞凋亡,呈现出典型细胞凋亡形态学变化。体内联合组同样能显著抑制肿瘤生长,瘤重抑瘤率达52.81％,免疫组化结果显示联合组能上调bax基因表达,同时下调bcl-2、VEGF等基因的表达。以上结果表明Ad-ING4-IL-24具有化疗增敏的作用,该作用机制可能与促进肿瘤细胞凋亡和抑制血管形成有关。     

Delivery of biologically active anti-inflammatory cytokines IL-10 and IL-1ra in vivo by the Shigella type III secretion apparatus

Pathogenicity of many Gram-negative bacteria relies on a type III secretion (T3S) apparatus, which is used for delivery of bacterial effectors into the host cell cytoplasm allowing the bacteria to manipulate host cell cytoskeleton network as well as to interfere with intracellular signaling pathways. In this study, we investigated the potential of the Shigella flexneri T3SA as an in vivo delivery system for biologically active molecules such as cytokines. The anti-inflammatory cytokines IL-10 and IL-1 receptor antagonist (IL-1ra) were genetically fused to the first 30 or 60 residues of the Shigella T3S effector IpaH9.8 or to the first 50 residues of the Yersinia enterocolitica effector YopE and the recombinant fusion proteins were expressed in S. flexneri. YopE(50)-IL-10, IpaH(60)-IL-10, and IpaH(60)-IL-1ra were efficiently secreted via the T3S apparatus of Shigella. Moreover, these recombinant proteins did not impair the invasive ability of the bacteria in vitro. In a murine model, Shigella strains expressing YopE(50)-IL-10, IpaH(60)-IL-10, and IpaH(60)-IL-1ra induced a lower mortality in mice that was associated with reduced inflammation and a restricted localization of bacteria within the lung tissues as compared with wild-type Shigella. Moreover, the level of TNF-alpha and IL-1beta mRNA were reduced in the lungs following infection by IL-10- and IL-1ra-secreting Shigella, respectively. These findings demonstrate that the Shigella T3S apparatus can deliver biologically active cytokines in vivo, thus opening new avenues for the use of attenuated bacteria to deliver proteins for immunomodulation or gene therapy purposes.     

IL-2与猪细小病毒VP2基因双表达载体的构建及免疫原性的研究

将白细胞介素-2基因和猪细小病毒VP2基因主要抗原区克隆至pCI-neo真核表达载体中,构建了pCIneo-IL2-VP2重组质粒,用脂质体将其转染到PK-15细胞中,利用免疫荧光方法检测在体外表达情况。并以小鼠为动物模型,将pCIneo-IL2-VP2重组质粒、对照组pCI-neo和猪细小病毒活疫苗通过肌肉注射进行免疫,检测免疫小鼠的淋巴细胞转化功能,特异性CTL杀伤活性和血清抗体滴度。结果显示,pCIneo-IL2-VP2在体外能够诱导PK-15细胞表达VP2蛋白,小鼠注射pCIneo-IL2-VP2质粒1周后能够诱导机体产生抗体,4周时达到峰值,与活疫苗对照组产生的抗体滴度、诱导T淋巴细胞增殖和诱导强的细胞毒性基本一致。试验表明,构建的pCIneo-IL2-VP2能够有效诱导机体产生体液免疫和细胞免疫。     

Releasable PEGylation of mesothelin targeted immunotoxin SS1P achieves single dosage complete regression of a human carcinoma in mice

Recombinant immunotoxins exhibit targeting and cytotoxic functions needed for cell-specific destruction. However, antitumor efficacy, safety, and pharmacokinetics of these therapeutics might be improved by further macromolecular engineering. SS1P is a recombinant anti-mesothelin immunotoxin in clinical trials in patients with mesothelin-expressing tumors. We have modified this immunotoxin using several PEGylation strategies employing releasable linkages between the protein and the PEG polymers, and observed superior performance of these bioconjugates when compared to similar PEG derivatives bearing permanent linkages to the polymers. PEGylated derivatives displayed markedly diminished cytotoxicity on cultured mesothelin-overexpressing A431-K5 cells; however, the releasable PEGylated immunotoxins exhibited increased antitumor activity in A431-K5 xenografts in mice, with a diminished animal toxicity. Most significantly, complete tumor regressions were achievable with single dose administration of the bioconjugates but not the native immunotoxin. Pharmacokinetic analysis of the releasable PEGylated derivatives in mice demonstrated an over 80-fold expansion of the area under the curve exposure of bioactive protein when compared to native immunotoxin. A correlation in degree of derivatization, release kinetics, and polymer size with potency was observed in vivo, whereas in vitro cytotoxicity was not predictive of efficacy in animal models. The potent antitumor efficacy of the releasable PEGylated mesothelin-targeted immunotoxins was not exhibited by similar untargeted PEG immunotoxins in this model. Since the bioconjugates can also exhibit the attributes of passive targeting via enhanced permeability and retention, this is the first demonstration of a pivotal role of active targeting for immunotoxin bioconjugate efficacy.     

IL-10_(23-57)-PE40可溶表达、纯化及其细胞活性

以IL-10的功能短肽(35肽,即IL10第23至57氨基酸残基)为导向部分与PE40(绿脓杆菌外毒素除去受体结合区后的剩余部分)融合分别置入pet20b(+)和pet28a(+)构建重组毒素IL102357PE40的两种表达质粒,其中置于pet20b(+)的重组毒素在BL21(DE3)pLysS中以周质分泌可溶形式表达,置于pet28a(+)的重组毒素在Rosettablue(DE3)中以高效胞质可溶形式表达;依次通过硫酸铵盐析、疏水层析、阴离子交换层析、铜离子亲和层析纯化周质分泌成份,得90%重组毒素纯品;细胞活性实验表明,该重组毒素只对单核巨噬细胞有杀伤作用;细胞ELISA显示,该重组毒素对单核巨噬细胞的杀伤作用(IC50为13.9pmolL)符合绿脓杆菌外毒素的作用机理.     

IL-1023-57-PE40可溶表达、纯化及其细胞活性

以IL-10的功能短肽(35肽,即IL10第23至57氨基酸残基)为导向部分与PE40(绿脓杆菌外毒素除去受体结合区后的剩余部分)融合分别置入pet20b( )和pet28a( )构建重组毒素IL102357PE40的两种表达质粒,其中置于pet20b( )的重组毒素在BL21(DE3)pLysS中以周质分泌可溶形式表达,置于pet28a( )的重组毒素在Rosettablue(DE3)中以高效胞质可溶形式表达;依次通过硫酸铵盐析、疏水层析、阴离子交换层析、铜离子亲和层析纯化周质分泌成份,得90%重组毒素纯品;细胞活性实验表明,该重组毒素只对单核巨噬细胞有杀伤作用;细胞ELISA显示,该重组毒素对单核巨噬细胞的杀伤作用(IC50为13.9pmolL)符合绿脓杆菌外毒素的作用机理.     

猪白细胞介素18成熟蛋白基因的克隆与表达

目的克隆猪白细胞介素18(pIL-18)成熟蛋白基因,并在植物乳杆菌(Lb.plantarum)NC8中进行表达。方法通过RT-PCR方法从猪脾脏细胞中扩增出pIL-18成熟蛋白基因,克隆到T载体pMD18-T后测序;将阳性基因片段克隆至大肠埃希菌-乳酸菌穿梭表达载体pSIP-409构建重组表达载体pSIP-409-IL-18,进行酶切和PCR鉴定;应用电穿孔技术将其转化至Lb.plantarum NC8中,经SppIP诱导表达后,进行SDS-PAGE及Western-blot分析。结果经测序,pIL-18成熟蛋白基因核苷酸长度为579 bp,编码193个核苷酸;酶切和PCR鉴定证明成功构建了重组表达载体pSIP-409-IL-18;SDS-PAGE及Western-blot分析表明重组菌表达了18 kD的融合蛋白,该重组蛋白可以与鼠抗猪IL-18多克隆抗体反应。结论成功克隆了pIL-18成熟蛋白基因,并获得有生物活性的pIL-18重组乳酸菌,为研制开发IL-18重组乳酸菌制剂奠定基础。     

小鼠白细胞介素33真核表达质粒的构建及表达

克隆小鼠IL-33基因构建其真核表达质粒,并转染COS-7细胞检测其表达。提取C57BL/6小鼠肺组织总RNA,经反转录聚合酶链式反应（RT-PCR）扩增小鼠IL-33基因,酶切后插入pcDNA^TM3.1/myc HisA构建其真核表达质粒pcDNA-3.1-IL-33,重组质粒转染COS-7细胞,RT-PCR和免疫印迹法（western blotting）检测目的基因表达。结果显示,pcDNA3.1-IL-33中插入的片段序列测定结果与小鼠IL-33cDNA序列一致,重组质粒转染COS-7细胞后检测到相应mRNA及蛋白表达。成功克隆了小鼠IL-33基因cDNA,并构建其真核表达质粒。     

IL-10 signaling is essential for 1,25-dihydroxyvitamin D3-mediated inhibition of experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) results from an aberrant, neuroantigen-specific, T cell-mediated autoimmune response. Because MS prevalence and severity decrease sharply with increasing sunlight exposure, and sunlight supports vitamin D(3) synthesis, we proposed that vitamin D(3) and 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) may protect against MS. In support of this hypothesis, 1,25-(OH)(2)D(3) strongly inhibited experimental autoimmune encephalomyelitis (EAE). This inhibition required lymphocytes other than the encephalitogenic T cells. In this study, we tested the hypothesis that 1,25-(OH)(2)D(3) might inhibit EAE through the action of IL-10-producing regulatory lymphocytes. We report that vitamin D(3) and 1,25-(OH)(2)D(3) strongly inhibited myelin oligodendrocyte peptide (MOG(35-55))-induced EAE in C57BL/6 mice, but completely failed to inhibit EAE in mice with a disrupted IL-10 or IL-10R gene. Thus, a functional IL-10-IL-10R pathway was essential for 1,25-(OH)(2)D(3) to inhibit EAE. The 1,25-(OH)(2)D(3) also failed to inhibit EAE in reciprocal, mixed bone marrow chimeras constructed by transferring IL-10-deficient bone marrow into irradiated wild-type mice and vice versa. Thus, 1,25-(OH)(2)D(3) may be enhancing an anti-inflammatory loop involving hemopoietic cell-produced IL-10 acting on brain parenchymal cells and vice versa. If this interpretation is correct, and humans have a similar bidirectional IL-10-dependent loop, then an IL-10-IL-10R pathway defect could abrogate the anti-inflammatory and neuro-protective functions of sunlight and vitamin D(3). In this way, a genetic IL-10-IL-10R pathway defect could interact with an environmental risk factor, vitamin D(3) insufficiency, to increase MS risk and severity.     

Improvement of a recombinant anti-monkey anti-CD3 diphtheria toxin based immunotoxin by yeast display affinity maturation of the scFv

Recently, a bivalent recombinant anti-human CD3 diphtheria toxin (DT) based immunotoxin derived from the scFv of UCHT1 antibody has been made that shows enhanced bioactivity and is free from the side effects of Fc receptor interaction. In this case, the diminution of CD3 binding due to the placement of the scFv domain at the C-terminus of the truncated DT in single scFv immunotoxins was compensated by adding an additional scFv domain. However, this strategy was less successful for constructing an anti-rhesus recombinant immunotoxin derived from the scFv of FN18 antibody due to poor binding of the anti-rhesus bivalent immunotoxin. We report here that, by increasing the FN18 scFv affinity through random mutagenesis and selection with a dye-labeled monkey CD3epsilongamma recombinant heterodimer, we greatly improved the bioactivity of FN18 derived immunotoxin. The best mutant, C207, contained nine mutations, two of which were located in CDRs that changed the charge from negative to positive. Binding affinity of the C207 scFv to the monkey T cell line HSC-F increased 9.8-fold. The potency of the C207 bivalent immunotoxin assayed by inhibition of protein synthesis increased by 238-fold.