Structure-activity profiles of Ab-derived TNF fusion proteins

TNF application in humans is limited by severe side effects, including life-threatening symptoms of shock. Therefore, TNF can be successfully applied as a tumor therapeutic reagent only under conditions that prevent its systemic action. To overcome this limitation, genetic fusion of TNF to tumor-selective Abs is a favored strategy to increase site-specific cytokine targeting. Because wild-type TNF displays its bioactivity as noncovalently linked homotrimer, the challenge is to define structural requirements for a TNF-based immunokine format with optimized structure-activity profile. We compared toxicity and efficacy of a dimerized CH2/CH3 truncated IgG1-TNF fusion protein and a single-chain variable fragment-coupled TNF monomer recognizing fibroblast-activating protein. The former construct preserves its dimeric structure stabilized by the natural disulfide bond IgG1 hinge region, while the latter trimerizes under native conditions. Analysis of complex formation of wild-type TNF and of both fusion proteins with TNFR type 1 (TNF-R1) using surface plasmon resonance correlated well with in vitro and in vivo toxicity data. There is strong evidence that TNF subunits in a trimeric state display similar toxicity profiles despite genetic fusion to single-chain variable fragment domains. However, LD(50) of either immunodeficient BALB/c nu/nu or immunocompetent BALB/c mice was significantly decreased following administration of TNF in the formation of IgG1-derived dimeric fusion protein. Reduction of unspecific peripheral complexation of TNF-R1 resulted in higher anticancer potency by immunotargeting of fibroblast-activating protein-expressing xenografts. The broader therapeutic window of the IgG1-derived TNF fusion protein favors the dimeric TNF-immunokine format for systemic TNF-based tumor immunotherapy.     

Recombinant 55-kDa tumor necrosis factor (TNF) receptor. Stoichiometry of binding to TNF alpha and TNF beta and inhibition of TNF activity.

The extracellular domain of the 55-kDa TNF receptor (rsTNFR beta) has been expressed as a secreted protein in baculovirus-infected insect cells and Chinese hamster ovary (CHO)/dhfr- cells. A chimeric fusion protein (rsTNFR beta-h gamma 3) constructed by inserting the extracellular part of the receptor in front of the hinge region of the human IgG C gamma 3 chain has been expressed in mouse myeloma cells. The recombinant receptor proteins were purified from transfected cell culture supernatants by TNF alpha- or protein G affinity chromatography and gel filtration. In a solid phase binding assay rsTNFR beta was found to bind TNF alpha with high affinity comparable with the membrane-bound full-length receptor. The affinity for TNF beta was slightly impaired. However, the bivalent rsTNFR beta-h gamma 3 fusion protein bound both ligands with a significantly higher affinity than monovalent rsTNFR beta reflecting most likely an increased avidity of the bivalent construct. A molecular mass of about 140 kDa for both rsTNFR beta.TNF alpha and rsTNFR beta.TNF beta complexes was determined in analytical ultracentrifugation studies strongly suggesting a stoichiometry of three rsTNFR beta molecules bound to one TNF alpha or TNF beta trimer. Sedimentation velocity and quasielastic light scattering measurements indicated an extended structure for rsTNFR beta and its TNF alpha and TNF beta complexes. Multiple receptor binding sites on TNF alpha trimers could also be demonstrated by a TNF alpha-induced agglutination of Latex beads coated with the rsTNFR beta-h gamma 3 fusion protein. Both rsTNFR beta and rsTNFR beta-h gamma 3 were found to inhibit binding of TNF alpha and TNF beta to native 55- and 75-kDa TNF receptors and to prevent TNF alpha and TNF beta bioactivity in a cellular cytotoxicity assay. Concentrations of rsTNFR beta-h gamma 3 equimolar to TNF alpha were sufficient to neutralize TNF activity almost completely, whereas a 10-100-fold excess of rsTNFR beta was needed for similar inhibitory effects. In view of their potent TNF antagonizing activity, recombinant soluble TNF receptor fragments might be useful as therapeutic agents in TNF-mediated disorders.     

Improved monovalent TNF receptor 1-selective inhibitor with novel heterodimerizing Fc

The development of alternative therapeutic strategies to tumor necrosis factor (TNF)-blocking antibodies for the treatment of inflammatory diseases has generated increasing interest. In particular, selective inhibition of TNF receptor 1 (TNFR1) promises a more precise intervention, tackling only the pro-inflammatory responses mediated by TNF while leaving regenerative and pro-survival signals transduced by TNFR2 untouched. We recently generated a monovalent anti-TNFR1 antibody fragment (Fab 13.7) as an efficient inhibitor of TNFR1. To improve the pharmacokinetic properties of Fab 13.7, the variable domains of the heavy and light chains were fused to the N-termini of newly generated heterodimerizing Fc chains. This novel Fc heterodimerization technology, designated “Fc-one/kappa” (Fc1κ) is based on interspersed constant Ig domains substituting the CH3 domains of a γ1 Fc. The interspersed immunoglobulin (Ig) domains originate from the per se heterodimerizing constant CH1 and CLκ domains and contain sequence stretches of an IgG1 CH3 domain, destined to enable interaction with the neonatal Fc receptor, and thus promote extended serum half-life. The resulting monovalent Fv-Fc1κ fusion protein (Atrosimab) retained strong binding to TNFR1 as determined by enzyme-linked immunosorbent assay and quartz crystal microbalance, and potently inhibited TNF-induced activation of TNFR1. Atrosimab lacks agonistic activity for TNFR1 on its own and in the presence of anti-human IgG antibodies and displays clearly improved pharmacokinetic properties.     

嵌合蛋白sTNFR II-IgG Fc的克隆、表达与活性分析

肿瘤坏死因子是一种重要的炎性细胞因子,目前已知许多免疫疾病与之相关,为了抑制TNF的生物学活性,将可溶性TNFR Ⅱ(sTNFR Ⅱ）和人IgG Fc分子通过柔性短肽相连,构建成一个嵌合蛋白,在大肠杆菌中进行表达,并获得了纯化蛋白。实验证明该嵌合蛋白能够自发形成聚合体,识别并结合TNF蛋白,同单体sTNFR Ⅱ相比,对TNF的中和活性得到了较大的提高。     

Selectivity of BAFF/BLyS and APRIL for binding to the TNF family receptors BAFFR/BR3 and BCMA

BAFF (B cell activating factor of the TNF family, also known as BlyS and TALL-1), a TNF family cytokine critical for the development and function of B cells, has been reported to bind to three receptors, BCMA (B cell maturation protein), TACI (transmembrane activator and CAML [calcium-modulator and cyclophilin ligand] interactor), and BAFFR (BAFF receptor), but with widely conflicting values for the affinity and selectivity of binding. BCMA and TACI additionally bind APRIL (a proliferation-inducing ligand), the TNF family ligand most homologous to BAFF. Using soluble, monomeric forms of the receptors, we demonstrate that BAFFR binds BAFF with K(D) approximately 16 nM, while BCMA binds with K(D) approximately 1.6 microM, indicating a approximately 100-fold selectivity for binding to BAFFR over BCMA. APRIL shows the opposite selectivity, binding to BCMA with K(D) approximately 16 nM while showing no detectable affinity for BAFFR (K(D) > 3 microM). The binding of BAFF or APRIL to these receptors is highly sensitive to assay-dependent avidity effects, likely explaining the widely ranging affinity values reported in the literature. Binding of BAFF to BCMA-Fc, a bivalent fusion protein consisting of the extracellular domain of BCMA fused to the hinge and CH1 and CH2 domains of human IgG1, in solution or coated onto an ELISA plate gave apparent binding affinities of approximately 0.63 and approximately 0.15 nM, respectively, compared to values of K(D(app)) 相似文献   

Biochemical characterization of a new recombinant TNF receptor-hyFc fusion protein expressed in CHO cells

The currently used Tumor Nectosis Factor (TNF)-α blockers such as infliximab, adalimumab and etanercept have Fc regions of the human IgG1 subtype have advantages in terms of in vivo half-life, however these could raise potential concerns for unwanted effector-mediated effects, such as antibody dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). To address this issue, we constructed a novel hybrid protein with decreased ADCC and CDC potentials by fusing the TNF receptor to a hybrid Fc (hyFc) containing CH2 and CH3 regions of IgG4 and highly flexible hinge regions of IgD which neither has ADCC and CDC activities. The resulting fusion protein, TNFR-hyFc, was over-expressed in CHO cells. For use as a pre-clinical material in pharmacology, PK and toxicological evaluations were carried out for biochemical characterization which was then compared with etanercept that has similarity in structure. Amino acid composition analysis and peptide mapping showed that the expressed TNFR-hyFc matched the theoretical composition derived from the DNA sequence. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) showed that TNFR-hyFc is 2.9 kDa larger than etanercept. MALDI-TOF after removal of N-glycans by PNGase treatment showed that TNFR-hyFc is 3.9 kDa larger than etanercept. Isoelectric focusing and monosaccharide analysis showed that TNFR-hyFc is slightly more acidic than etanercept. N-terminal amino acid sequencing showed that N-terminal heterogeneity is present in both TNFR-hyFc and etanercept, although the ratios are somewhat different. Glycan analysis showed that the main glycan form is bi-antennary, similar to etanercept.     

The pharmacology study of a new recombinant TNF receptor-hyFc fusion protein

TNF-α-blocking agents such as infliximab, adalimumab and etanercept are widely used for the treatment of severe inflammatory diseases including rheumatoid arthritis and psoriasis. The currently used TNF-α blockers have Fc regions of the human IgG1 subtype, which is advantageous in terms of in vivo half-life but also raise the potential for unwanted effector-mediated effects, such as antibody dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). To address this issue, we constructed a novel hybrid protein by fusing the TNF receptor (TNFR) with a hybrid Fc (hyFc) consisting of the CH2 and CH3 regions of IgG4 and the highly flexible hinge regions of IgD which would not have ADCC and CDC activity. The resulting fusion protein, TNFR-hyFc, was over-expressed in CHO and pharmacological characteristics were evaluated in comparison with the structurally similar etanercept. TNFR-hyFc effectively neutralized TNF-α in L929 bioassay and showed a 1.5-fold higher neutralizing activity compared to etanercept. In a pharmacokinetic study in cynomolgus monkeys, TNFR-hyFc showed plasma half-life and AUC comparable to etanercept. In a mouse collagen induced arthritis model, TNFR-hyFc showed significant amelioration of arthritis compared to etanercept or vehicle control. In an LPS-induced septic shock model, TNFR-hyFc showed a similar level of protection against mortality as etanercept. These results confirm the feasibility of the TNFR-hyFc as an effective TNF-α blocker for the treatment of inflammatory diseases.     

Superior serum half life of albumin tagged TNF ligands

Due to their immune stimulating and apoptosis inducing properties, ligands of the TNF family attract increasing interest as therapeutic proteins. A general limitation of in vivo applications of recombinant soluble TNF ligands is their notoriously rapid clearance from circulation. To improve the serum half life of the TNF family members TNF, TWEAK and TRAIL, we genetically fused soluble variants of these molecules to human serum albumin (HSA). The serum albumin-TNF ligand fusion proteins were found to be of similar bioactivity as the corresponding HSA-less counterparts. Upon intravenous injection (i.v.), serum half life of HSA-TNF ligand fusion proteins, as determined by ELISA, was around 15 h as compared to approximately 1 h for all of the recombinant control TNF ligands without HSA domain. Moreover, serum samples collected 6 or 24 h after i.v. injection still contained high TNF ligand bioactivity, demonstrating that there is only limited degradation/inactivation of circulating HSA-TNF ligand fusion proteins in vivo. In a xenotransplantation model, significantly less of the HSA-TRAIL fusion protein compared to the respective control TRAIL protein was required to achieve inhibition of tumor growth indicating that the increased half life of HSA-TNF ligand fusion proteins translates into better therapeutic action in vivo. In conclusion, our data suggest that genetic fusion to serum albumin is a powerful and generally applicable mean to improve bioavailability and in vivo activity of TNF ligands.     

In vivo tumor delivery of a recombinant single chain Fv::tumor necrosis factor-alpha fusion [correction of factor: a fusion] protein.

Locoregional and intratumoral administration of tumor necrosis factor alpha (TNF alpha) has been successful in obtaining inhibition or regression of tumor growth in the clinic. This potent antitumor activity of TNF alpha has not yet been exploited as a systemic agent in cancer therapy, mainly due to high levels of toxicity to normal tissues before a therapeutic dose of TNF alpha in the tumor has been achieved. To address this, we have targeted TNF alpha using antitumor antibodies. We have used a genetic fusion of human recombinant TNF alpha with MFE-23, a single-chain Fv antibody fragment directed against carcinoembryonic antigen. MFE-23::TNF alpha fusion protein is isolated in high yields (28 mg/L) from bacterial inclusion bodies and purified to homogeneity by affinity chromatography. It is a 144 kDa trimer in native form and possesses the antigen-binding activity of the sFv and the cytotoxicity to both WEHI 164 and a human adenocarcinoma cell line (LoVo) of rhTNF alpha. Radiolabeled MFE-23::TNF alpha binds both human and mouse TNF receptor 1 in vitro and is able to localize effectively in nude (nu/nu) mice bearing human LS174T xenografts; tumor/tissue ratios of 21:1 and 60:1 are achieved 24 and 48 h after intravenous injection. These studies indicate that MFE-23::TNF alpha will provide an effective means for systemically administered cancer therapy with TNF alpha.     

炭疽毒素受体与人IgG1的Fc段融合蛋白ATR_Fc在CHO细胞中的表达

ATR_Fc是人炭疽毒素受体(ATR)的胞外区与人免疫球蛋白IgG1的铰链区、CH2区和CH3区组成的融合蛋白。表达该蛋白是为了获得结合PA的抗体样分子,通过阻断PA与细胞受体的结合,而阻止炭疽致死毒素和水肿因子进入细胞内,可作为预防和治疗炭疽感染的生物制品。将编码炭疽毒素受体N端1_227氨基酸的基因和编码Fc段的基因连接,插入到pcDNA3.1的HindⅢ和NotⅠ位点得到表达ATR_Fc融合蛋白的真核表达载体pcDNA3.1ATR_Fc,并用脂质体方法将该载体转染至CHO_K1细胞中,用G418筛选并获得ATR_Fc表达水平为10～15μg(106cells·d)的基因工程CHO细胞系ATR_Fc_1D5。采用蛋白A纯化重组蛋白,并用ELISA法鉴定ATR_Fc与PA的亲和性,表明ATR_Fc可与PA特异性结合。     

人可溶性肿瘤坏死因子受体Ⅱ与IgG Fc融合蛋白在乳酸克鲁维酵母菌中的表达及产物分析

目的:在乳酸克鲁维酵母中表达人可溶性肿瘤坏死因子受体Ⅱ(sTNFRⅡ)与IgG Fc的融合蛋白。方法:首先获得sTNFRⅡ-IgGFc融合基因片段,然后构建至乳酸克鲁维酵母表达载体pKLAC1中,获得sTNFRⅡ-IgGFc的表达载体,并将其电转化乳酸克鲁维酵母(Δura3),通过ELISA方法筛选高表达sTNFRⅡ-IgGFc融合蛋白的重组乳酸克鲁维酵母菌株,采用还原和非还原SDS-PAGE分析融合蛋白是否形成二聚体结构,Western印迹验证sTNFRⅡ-IgGFc融合蛋白在乳酸克鲁维酵母(Δura3)中的表达。结果:构建了sTNFRⅡ-IgGFc表达载体pKLAC1-sTNFRⅡ-IgGFc,获得了表达sTNFRⅡ-IgGFc的乳酸克鲁维酵母菌株,SDS-PAGE和Western印迹表明该融合蛋白能自发形成类似于抗体的二聚体结构。结论:实现了sTNFRⅡ-IgGFc融合蛋白在乳酸克鲁维酵母(Δura3)中的表达。     

TNF receptor-associated factor 5 limits the induction of Th2 immune responses

The TNF receptor-associated factor (TRAF) family of molecules acts as adapter proteins for signaling pathways initiated by several members of the TNF receptor (TNFR) superfamily. TRAF5(-/-) animals are viable and have normal development of the immune system despite interacting with several TNFR family members. A clear role for TRAF5 has yet to emerge. OX40 (CD134) interacts with TRAF5, suggesting that this pathway could be involved in regulating T cell differentiation into Th1 or Th2 cells. In tissue culture, OX40 stimulation of TRAF5(-/-) T cells resulted in a pronounced Th2 phenotype with elevated levels of IL-4 and IL-5. Similarly, in vivo immunization with protein in adjuvant in the presence of an agonist anti-OX40 Ab resulted in enhanced Th2 development in TRAF5(-/-) mice. Additionally, lung inflammation induced by T cells, which is critically controlled by OX40, was more pronounced in TRAF5(-/-) mice, characterized by higher levels of Th2 cytokines. These results suggest that TRAF5 can limit the induction of Th2 responses, and that TRAF5 can play a role in modulating responses driven by OX40 costimulation.     

Seeing double: Crystal structures of the type I TNF receptor

The crystal structure of the extracellular domain of the type I tumor necrosis factor receptor (sTNF-R1) has been determined to 2.25 Å at pH 7.5. We have also solved the structure of sTNF-R1 at pH 3.7. sTNF-R1 is an elongated molecule consisting of a linear combination of four cysteine-rich motifs. Interestingly, the crystal structure reveals two distinct dimers of the receptor. One dimer is formed by a parallel arrangement of receptors, the other by an antiparallel arrangement of receptors. In the parallel arrangement of the receptors, the tumor necrosis factor (TNF) binding face of the receptor is completely exposed to solvent. However, in the antiparallel arrangement, the TNF binding face is intimately involved in the dimer interactions. Details of these recognition surfaces are discussed. Both these dimer interactions bury substantial surface area, comprise polar and apolar contact surfaces and have complimentary recognition surfaces. Thus these interactions are typical of genuine protein–protein interactions, rather than crystal packing contacts. These dimers may function to inhibit signal transduction in the absence of TNF or in the case of the parallel dimer, promote clustering of TNF/TNF receptor complexes on the cell surface.     

Effect of different tumor necrosis factor (TNF) reactive agents on reverse signaling of membrane integrated TNF in monocytes

Reverse signaling of transmembrane TNF (mTNF) contributes to the versatility of this cytokine superfamily. Previously, we could demonstrate that mTNF acting as receptor confers resistance to bacterial lipopolysaccharide in monocytes and macrophages (MO/MPhi). Reverse signaling can be induced by incubation with the monoclonal anti-TNF antibody 195F and other TNF antagonists, such as the humanized monoclonal antibody infliximab and the humanized soluble TNF receptor construct etanercept, respectively, all in former or present clinical use. Here, we addressed the question whether there are differences in modulating the LPS response in MO/MPhi among these three antagonists. Whereas 195F and infliximab suppress both, the release of an LPS-induced endothelial cell apoptotic factor and proinflammatory cytokines, etanercept only protected against the LPS-triggered apoptosis activity, but left the LPS-induced cytokine release unchanged. These data could have clinical impact with regard to TNF neutralization strategies.     

Enhancement of cell-mediated cytotoxicity by recombinant tumor necrosis factor (TNF alpha)

The effects of recombinant tumor necrosis factor (rTNF alpha) on the immune responses were investigated. A single iv injection of rTNF alpha (6 x 10(3) U) caused regression of sarcoma-180 transplanted into BALB/c nu/+ mice, but failed to regress this tumor in nu/nu mice. A higher dose of rTNF alpha (2 x 10(4) U) was necessary to induce antitumor effect in nu/nu mice. A host-related factor seemed to be involved in mediating tumor regression. Therefore, the effects of rTNF alpha on various T-dependent immune responses, including delayed footpad reaction (DFR), cell mediated cytolysis (CMC), and plaque-forming cells (PFC) were examined in BALB/c mice, immunized ip with chicken erythrocytes (CRBC). A single injection of rTNF alpha, at the time of the antigen administration, induced the augmentation of CMC to CRBC in a dose-dependent manner. DFR and PFC were not affected in optimal immunization procedures. The TNF alpha injection, at or after the time of antigen administration, was more effective in inducing augmentation of CMC. The increase in CMC by TNF alpha was mediated by nonadherent, Thy 1.2, Lyt 2.2 positive cells and neutralization of TNF alpha by the anti-TNF alpha monoclonal antibody abolished the effect on CMC. These results indicated that the human recombinant TNF alpha induced changes in the T-cell-mediated responses.     

A Novel Bifunctional Protein TNFR2-Fc-IL-1ra (TFI): Expression, Purification and its Neutralization Activity of Inflammatory Factors

Tumor necrosis factor receptor (TNF) and internleukin-1 (IL-1) are the most potent proinflammatory cytokines involving in autoimmune and inflammatory human diseases. Many anti-inflammatory agents have been exploited for anti-inflammation treatments by targeting cytokines including TNF and IL-1. Theoretically, simultaneously neutralizing or blocking two important inflammatory mediators may achieve a synergistic therapeutic effect. We have developed a recombinant fusion protein, TNFR2-Fc-IL-1ra (TFI), which consists of a TNF-neutralizing domain that specifically binds to TNF-α, an IL-1 receptor antagonist domain, and a dimerization Fc portion of human IgG1, for bifunctional inflammatory inhibitor. Recombinant DNA expressing the sequence of this fusion protein was expressed in CHO-S cells. The protein product was purified using a two-step purification protocol and the identity of the protein was confirmed by western blot analysis. The purified recombinant protein had a purity of about 98 % as determined by HPLC, and a molecular mass of 164.6 kDa as determined by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The results of cell binding inhibition indicate that TFI was able to strongly neutralize TNF activity and antagonize IL-1r activity, suggesting that TFI may be used as a bifunctional ligand with enhanced anti-inflammatory effect. The result obtained in this study may provide a platform for extending bifunctional anti-inflammatory drug development.     

ATROSAB,a humanized antagonistic anti-tumor necrosis factor receptor one-specific antibody

Tumor necrosis factor (TNF) signals through TNFR1 and TNFR2, two membrane receptors, and TNFR1 is known to be the major pathogenic mediator of chronic and acute inflammatory diseases. Present clinical intervention is based on neutralization of the ligand TNF. Selective inhibition of TNF receptor 1 (TNFR1) provides an alternative opportunity to neutralize the pro-inflammatory activity of TNF while maintaining the advantageous immunological responses mediated by TNFR2, including immune regulation, tissue homeostasis and neuroprotection. We recently humanized a mouse anti-human TNFR1 monoclonal antibody exhibiting TNFR1-neutralizing activity. This humanized antibody has been converted into an IgG1 molecule (ATROSAB) containing a modified Fc region previously demonstrated to have greatly reduced effector functions. Purified ATROSAB produced in CHO cells showed strong binding to human and rhesus TNFR1-Fc fusion protein and mouse embryonic fibroblasts transfected with a recombinant TNFR1 fusion protein with an affinity identical to the parental mouse antibody H398. Using chimeric human/mouse TNFR1 molecules, the epitope of ATROSAB was mapped to the N-terminal region (amino acid residues 1–70) comprising the first cysteine-rich domain (CRD1) and the A1 sub-domain of CRD2. In vitro, ATROSAB inhibited typical TNF-mediated responses like apoptosis induction and activation of NFκB-dependent gene expression such as IL-6 and IL-8 production. These findings open the way to further analyze the therapeutic activity of ATROSAB in relevant disease models in non-human primates.Key words: humanized IgG, antagonistic antibody, tumor necrosis factor receptor 1, epitope mapping     

Recovery of choline oxidase activity by in vitro recombination of individual segments

Elevated levels of B cell-activating factor of the TNF family (BlyS) have been implicated in the pathogenesis of autoimmune diseases in human. Removal of pathogenic B lymphocytes by decoy receptors has demonstrated clinical benefit in both oncological and immunological diseases. In this report, we have constructed vectors for the convenient and rapid expression of the extracellular domain of BR3(sBR3) fused to the Fc fragment (hinge, CH2, CH3) of human IgG1 in the methylotrophic yeast, Pichia pastoris. SDS-PAGE assays of culture broth from a methanol-induced expression strain demonstrated that the recombinant sBR3-Fc fusion protein is secreted and recovered from the culture medium as a disulfide-linked, glycosylated homodimer. The recombinant protein was purified to >95% using protein A affinity chromatography and size exclusion chromatography steps. Bioactivity of the recombinant sBR3-Fc was confirmed by the ability of the protein to inhibit mouse B lymphocyte proliferation induced by BLyS in vitro. Our results suggest that the P. pastoris expression system can be used to produce large quantities of fully functional sBR3-Fc fusion protein for both research and industrial purposes.     

Human immunodeficiency viral protease is catalytically active as a fusion protein: characterization of the fusion and native enzymes produced in Escherichia coli

Processing of the gag and pol gene precursor proteins of retroviruses is essential for the production of mature infectious virions. The processing is directed by a viral protease that itself is part of these precursors and is presumed to cleave itself autocatalytically. To facilitate study of this process, the protease was produced as a fusion protein in Escherichia coli. In this construct, the 10,793-Da protease was preceeded by two copies of a modified IgG binding domain derived from protein A. The IgG binding domain was linked to the protease by an Asp-Pro peptide bond which could not be cleaved by the viral protease. A dimer of the 25,400-Da fusion protein was catalytically active, specifically cleaving a substrate peptide at the correct Tyr-Pro bond. Thus, the fusion protein could serve as a model of the viral gag-pol polyprotein. The finding that the fusion protein was catalytically active supports the suggestion that a gag-pol dimer can initiate a proteolytic cascade after budding of the immature virus. The fusion protein also provided a source of authentic protease. The protease was released from the fusion construct by incubation with formic acid, cleaving the Asp-Pro linkage which had been inserted between the IgG binding domain and the protease.     

A single-chain IL-12 IgG3 antibody fusion protein retains antibody specificity and IL-12 bioactivity and demonstrates antitumor activity.

IL-12 is a heterodimeric cytokine with many actions on innate and cellular immunity that may have antitumor and antimetastatic effects. However, systemic administration of IL-12 can be toxic. Tumor-specific Abs provide a means to selectively target a metastatic/residual nodule and deliver therapeutic quantities of an immunostimulatory molecule like IL-12 with lower systemic levels and ideally, toxicity. We report the construction and characterization of an Ab fusion protein in which single-chain murine IL-12 is fused to an anti-Her2/neu Ab at the amino terminus (mscIL-12.her2.IgG3). The use of single-chain IL-12 in the fusion protein simplifies vector construction, ensures equimolar concentrations of the two IL-12 subunits, and may confer greater stability to the fusion protein. SDS-PAGE analysis shows this 320-kDa protein is secreted and correctly assembled. FACS analysis demonstrates that this fusion protein binds to cells transfected with the Her2/neu Ag, thus retaining Ab specificity; this fusion protein also binds to a cell line and to PHA-activated PBMC that express the IL-12R, thus demonstrating cytokine receptor specificity. T cell proliferation assays and NK cytotoxicity assays demonstrate that this fusion protein exhibits IL-12 bioactivity comparable to recombinant murine IL-12. In vivo studies demonstrate that this fusion protein has antitumor activity. These results are significant and suggest that this IL-12 Ab fusion protein can effectively combine the therapeutic potential of IL-12 with the tumor-targeting ability of the Ab and may provide a viable alternative to systemic administration of IL-12.