Development of TNKase-specific cleavable peptide-linked radioimmunoconjugates for radioimmunotherapy

Radioimmunotherapy (RIT) is a method for selectively delivering radionuclides to cancer cells while reducing the radiation dose to normal tissues. However, because of slow clearance of MAbs, normal tissues also received radiotoxicity. One of the promising strategies is linking on-demand cleavable (ODC) peptides between radiometal chelates and the tumor targeting agents. We have tested this proof-of-concept by using ODC peptides that are designed to be cleaved only by TNKase and are resistant to cleavage by enzymes present in the plasma and the tumor. TNKase-specific peptide linkers using l- and d-amino acids were screened by OBOC combinatorial peptide libraries. One of the best peptides was linked to radiometal chelate and ChL6-MAb to prepare radioimmunoconjugate (RIC). Optimization and characterization of the linker conjugation to MAb show (a) 1–2 peptides linked to each MAb; (b) immunoreactivity >80%; (c) specific activity of the RIC 0.7–1 μCi/μg; (d) RIC stable over 7 days in human plasma; and (e) radiometal-chelated ODC peptide cleaved from the RIC in plasma by TNKase at clinical dose levels of 10 μg/ml. The percent release of radiochelate from RIC was 50% at 24 h and 85% over 72 h in vitro. This novel ODC-linked RIC could be a potential molecule for RIT.     

Tailoring protease-sensitive photodynamic agents to specific disease-associated enzymes

We have developed novel polymeric photosensitizer prodrugs (PPPs) for improved photodynamic therapy. In PPPs, multiple photosensitizer units are covalently coupled to a polymeric backbone via protease-cleavable peptide linkers. These initially non-photoactive compounds become fluorescent and phototoxic after specific enzymatic cleavage of the peptide linkers and subsequent release of the photosensitizer moieties. Tethering the photosensitizer via a short and easily modified amino acid sequence to the polymeric backbone allows for the targeting of a wide variety of proteases. Model compounds, sensitive to trypsin-mediated cleavage, with different pheophorbide a-peptide loading ratios and backbone net charges were evaluated with respect to their solubility, "self-quenching" capacity of fluorescence emission, and reactive oxygen species (ROS) generation. In addition, linker sequence impaired selectivity toward enzymatic cleavage was demonstrated either by incubating PPPs with different enzymes having trypsin-like activity or by introducing a single d-arginine mutant in the peptide sequence. In vitro cell culture tests confirmed dose-dependent higher phototoxicity of enzymatically activated PPPs compared to the nonactivated conjugate after irradiation with white light. These data suggest that similar compounds adapted to disease-associated proteases can be used for selective photodynamic therapy.     

Precise nucleotide sequence modifications with bidirectionally cleaving class-IIS excision linkers

Bidirectionally cleaving blunt-ended DNA linkers have been constructed to generate defined nucleotide sequence modifications. The oligodeoxynucleotides (termed 'excision linkers'), contain two back-to-back recognition sites for class-IIS restriction endonucleases and provide a new instrument for modifying DNA primary structure. Following insertion of these linkers into host DNA, digestion with the cognate class-IIS enzyme results in a cleavage upstream and downstream from the adjoining enzyme recognition sites. Bidirectional cleavage efficiency can be improved by including spacer nucleotides between the two recognition sites. The number of nucleotides removed from or added to the host DNA depends upon the cleavage shift characteristic of the class-IIS enzyme, the design of the linker (including lateral spacer nucleotides to set the cleavage position), and the method used to make blunt ends from staggered ends following excision of the linker. BspMI linkers constructed in this study have been used to generate defined deletions in the ApR and TcR genes of pBR322. BsmI excision linkers are also described.     

重组单链抗体-低分子质量尿激酶融合蛋白在 CHO 细胞中的抗降解研究

抗人纤维蛋白单链抗体-低分子质量尿激酶(Ⅱn-UK)融合蛋白,兼有单链抗体对纤维蛋白的亲和性和尿激酶的溶栓活性,有望开发成为新型导向溶栓药物.但基于通用连接肽(G4S)3的Ⅱn-linker-UK融合蛋白在CHO细胞中表达时出现明显的降解.为了解决此问题,利用分子生物学方法,对Hn-UK融合蛋白进行了分子改造,包括置换连接肽,改变两个半分子(moiety)的相对位置,以及对连接肽附近明确的蛋白酶位点进行突变等方法,并分别研究了改造后的11种Ⅱn-1inker-UK或UK-linker-Ⅱn突变体在CHO细胞中分泌性表达时的稳定性,最终筛选到一种抗降解的突变体.     

Influence of the linker on the biodistribution and catabolism of actinium-225 self-immolative tumor-targeted isotope generators

Current limitations to applications of monoclonal antibody (mAb) targeted isotope generators in radioimmunotherapy include the low mAb labeling yields and the nonspecific radiation of normal tissues by nontargeted radioimmunoconjugates (RIC). Radiotoxicity occurs in normal organs that metabolize radiolabeled proteins and peptides, primarily liver and kidneys, or in radiosensitive organs with prolonged exposure to the isotope from the blood, such as the bone marrow. Actinium-225 nanogenerators also have the problem of released agar-emitting daughters. We developed two new bifunctional chelating agents (BCA) in order to address these issues. Thiol-maleimide conjugation chemistry was employed to increase the efficiency of the mAb radiolabelings by up to 8-fold. In addition, one bifunctional chelating agent incorporated a cleavable linker to alter the catabolism of the alpha-particle-emitting mAb conjugate. This linker was designed to be sensitive to cathepsins to allow release and clearance of the chelated radiometal after internalization of the radioimmunoconjugate into the cell. We compared the properties of the cleavable conjugate (mAb-DOTA-G3FC) to noncleavable constructs (mAb-DOTA-NCS and mAb-DOTA-SH). The cleavable RIC was able to release 80% of its radioactive payload when incubated with purified cathepsin B. The catabolism of the constructs mAb-DOTA-G3FC and mAb-DOTA-NCS was investigated in vitro and in vivo. RIC integrity was retained at 85% over a period of 136 h in mouse serum in vivo. Both conjugates were degraded over time inside HL-60 cells after internalization and in mouse liver in vivo. While we found that the rates of degradation of the two RICs in those conditions were similar, the amounts of the radiolabeled product residues were different. The cleavable mAb-DOTA-G3FC conjugate yielded a larger proportion of fragments below 6kDa in size in mouse liver in vivo after 12 h than the DOTA-NCS conjugate. Biodistribution studies in mice showed that the mAb-DOTA-G3FC construct yielded a higher liver dose and prolonged liver retention of radioactivity compared to the mAb-DOTA-NCS conjugate. The accumulation in the liver seemed to be in part caused by the maleimide functionalization of the antibody, since the noncleavable mAb-DOTA-SH maleimide-functionalized control conjugate displayed the same biodistribution pattern. These results provide an insight into the catabolism of RICs, by demonstrating that the release of the radioisotope from a RIC is not a sufficient condition to allow the radioactive moiety to clear from the body. The excretion mechanisms of radiolabeled fragments seem to constitute a major limiting step in the chain of events leading to their clearance.     

Modulation of single-chain antibody affinity with temperature-responsive elastin-like polypeptide linkers

Single-chain antibodies are genetically engineered constructs composed of a VH and VL domain of an antibody linked by a flexible peptide linker, commonly (GGGGS)3. We asked whether replacement of this flexible linker with peptides known to undergo environmentally induced structural transitions could lead to antibodies with controlled binding and release characteristics. To this end, we genetically modified and produced a series of anti-fluorescein single-chain antibodies with the general linker sequence (VPGXG)n, where n is 1.2 to 3 and X is Val or His, to evaluate the effects of linker length and composition. Our results indicate that single-chain antibodies containing elastin-like polypeptide linkers have equilibrium affinity (KD) comparable to wild-type (GGGGS)3 at room temperature but altered binding kinetics and faster ligand release as the temperature is raised. These results are consistent with the increased molecular order and contraction that elastin-like polypeptides are known to undergo with increased temperature. Modulation of antibody affinity using stimulus-responsive linkers may have applications in biosensors, drug delivery, and bioseparations.     

Development of albumin-binding camptothecin prodrugs using a Peptide positional scanning library

Designing truly tumor-specific prodrugs remains a challenge in the field of cancer chemotherapy. As a new strategy, we incubated homogenates of a spectrum of human colon tumor xenografts with a fluorogenic positional scanning tetrapeptide library in order to identify peptide sequences that are preferentially cleaved by colon tumors. Our screening experiments at pH 7.4 revealed that Met, Leu, and Lys were preferred amino acids in the position P(1) and Tyr, Phe, and Met in P(2), whereas in P(3) and P(4), the cleavage profiles were less characteristic. However, similar results were obtained when testing breast tumor material and homogenates from healthy murine organs. On the basis of these results, we developed albumin-binding camptothecin (CPT) prodrugs of the general formula EMC-Arg-P(4)-P(3)-P(2)-P(1)-Ala-CPT (EMC = 6-maleimidocaproic acid) that incorporated two peptide linkers: H-Arg-Ala-Phe-Met-OH and H-Arg-Phe-Tyr-Met-OH (P(4)-P(3)-P(2)-P(1)). The incorporation of two arginine residues rendered the prodrugs water-soluble (>7 mg/mL), while the use of alanine as an amino acid spacer proved to be beneficial for the release of the active agent. Incubation studies with homogenates of HT-29 colon tumor tissue and murine spleen, liver, and kidneys demonstrated cleavage of the peptide linker with CPT-peptide derivatives and CPT being the major cleavage products. Although the peptide sequence is not selectively cleaved in colon tumors, an in vivo study in a HT-29 xenograft model showed that the prodrug EMC-Arg-Arg-Ala-Phe-Met-Ala-CPT demonstrated enhanced antitumor efficacy when compared to CPT [( T/ C max: 17% for the prodrug (2 x 12.5 mg/kg CPT equivalents) and 40% for CPT (3 x 12.5 mg/kg)].     

Synthesis and biological in vitro evaluation of novel PEG-psoralen conjugates

Psoralens are well-known photosensitizers, and 8-methoxypsoralen and 4,5',8-trimethylpsoralen are widely used in photomedicine as "psoralens plus UVA therapy" (PUVA), in photopheresis, and in sterilization of blood preparations. In an attempt to improve the therapeutic efficiency of PUVA therapy and photopheresis, four poly(ethylene glycol) (PEG)-psoralen conjugates were synthesized to promote tumor targeting by the enhanced permeability and retention (EPR) effect. Peptide linkers were used to exploit specific enzymatic cleavage by lysosomal proteases. A new psoralen, 4-hydroxymethyl-4',8-dimethylpsoralen (6), suitable for polymer conjugation was synthesized. The hydroxy group allowed exploring different strategies for PEG conjugation, and linkages with different stability such ester or urethanes were obtained. PEG (5 kDa) was covalently conjugated to the new psoralen derivative using four different linkages, namely, (i) direct ester bond (7), (ii) ester linkage with a peptide spacer (8), (iii) a carbamic linker (9), and (iv) a carbamic linker with a peptide spacer (12). The stability of these new conjugates was assessed at different pHs, in plasma and following incubation with cathepsin B. Conjugates 7 and 8 were rapidly hydrolyzed in plasma, while 9 was stable in buffer and in the presence of cathepsin B. As expected, only the conjugates containing the peptide linker released the drug in presence of cathepsin B. In vitro evaluation of the cytotoxic activity in the presence and absence of light was carried out in two cell lines (MCF-7 and A375 cells). Conjugates 7 and 8 displayed a similar activity to the free drug (probably due to the low stability of the ester linkage). Interestingly, the conjugates containing the carbamate linkage (9 and 12) were completely inactive in the dark (IC50 > 100 microM in both cell lines). However, antiproliferative activity become apparent after UV irradiation. Conjugate 12 appears to be the most promising for future in vivo evaluation, since it was relatively stable in plasma, which should allow tumor targeting and drug release to occur by cathepsin B-mediated hydrolysis.     

Influences of hydrocarbon linkers on the receptor binding affinities of gonadotropin-releasing hormone peptides

Three new DOTA-conjugated GnRH peptides with various hydrocarbon linkers were synthesized to evaluate the influences of the linkers on their receptor binding affinities. The hydrocarbon linker displayed a profound impact on the receptor binding affinities of DOTA-conjugated GnRH peptides. The Aun linker was better than Gaba, Ahx and Aoc linkers in retaining strong receptor binding affinity of the GnRH peptide. DOTA-Aun-(d-Lys⁶-GnRH) displayed 22.8 nM GnRH receptor binding affinity. ¹¹¹In-DOTA-Aun-(d-Lys⁶-GnRH) exhibited fast tumor uptake and urinary clearance in MDA-MB-231 human breast cancer-xenografted nude mice. The cellular and biological results provided an insight into the design of new GnRH peptides in the future.     

Conditionally cleavable radioimmunoconjugates: a novel approach for the release of radioisotopes from radioimmunoconjugates

One of the limitations of therapy with radiolabeled monoclonal antibodies (mAbs) is that significant toxicities can arise from circulating non-tumor-bound radiolabeled conjugate. Here, we describe a new method to reduce systemic radiation exposure from radiolabeled mAbs involving the attachment of the radioisotope through a linker that can be cleaved by an administered enzyme. To demonstrate the feasibility of this approach, we prepared a conditionally cleavable radioimmunoconjugate (RIC) composed of (131)I-labeled cephalosporin conjugated to Tositumomab, a mAb against the CD20 antigen. The cleavable RIC bound antigen identically to directly iodinated antibody, and in the presence of beta-lactamase, about 80-85% of the radioisotope was released. In vivo studies in mice revealed that the cleavable RIC and the directly iodinated anti-CD20 antibody had similar biodistribution patterns. Systemically administered beta-lactamase induced a 2-3-fold decrease in the percent injected dose (ID) of the cleavable RIC/g of blood, marrow, spleen, lung, and liver 1 h after enzyme treatment, and a 4-6-fold decrease 20 h after enzyme treatment. This was accompanied by a 20-fold increase in % ID/g in urine 1 h after enzyme treatment, indicating that the released radiolabel was rapidly excreted through the kidneys. In mice with human tumor xenografts, there was no decrease in the %ID/g in tumor 1 h after enzyme treatment, but by 4 h after enzyme injection, decreases in tumor radioactive content began to diminish the targeting advantage. These studies demonstrate that the cleavable RIC substrate is able to bind to tumor antigens and localize within human tumor xenografts and that accelerated systemic clearance can be induced with beta-lactamase.     

Novel peptide linkers for highly potent antibody-auristatin conjugate

Auristatins are highly potent antimitotic agents that have received considerable attention because of their activities when targeted to tumor cells in the form of antibody-drug conjugates (ADCs). Our lead agent, SGN-35, consists of the cAC10 antibody linked to the N-terminal amino acid of monomethylauristatin E (MMAE) via a valine-citrulline p-aminobenzylcarbamate (val-cit-PABC) linker that is cleaved by intracellular proteases such as cathepsin B. More recently, we developed an auristatin F (AF) derivative monomethylauristatin F (MMAF), which unlike MMAE contains the amino acid phenylalanine at the C-terminal position. Because of the negatively charged C-terminal residue, the potency of AF and MMAF is impaired. However, their ability to kill target cells is greatly enhanced through facilitated cellular uptake by internalizing mAbs. Here, we explore the effects of linker technology on AF-based ADC potency, activity, and tolerability by generating a diverse set of dipeptide linkers between the C-terminal residue and the mAb carrier. The resulting ADCs differed widely in activity, with some having significantly improved therapeutic indices compared to the original mAb-Val-Cit-PABC-MMAF conjugate. The therapeutic index was increased yet further by generating dipeptide-based ADCs utilizing new auristatins with methionine or tryptophan as the C-terminal drug residue. These results demonstrate that manipulation of the C-terminal peptide sequence used to attach auristatins to the mAb carrier can lead to highly potent and specific conjugates with greatly improved therapeutic windows.     

Antisense oligonucleotide containing an internal, non-nucleotide-based linker promote site-specific cleavage of RNA.

We have designed and synthesized a series of novel antisense methylphosphonate oligonucleotide (MPO) cleaving agents that promote site-specific cleavage on a complementary RNA target. These MPOs contain a non- nucleotide-based linking moiety near the middle of the sequence in place of one of the nucleotide bases. The region surrounding the unpaired base on the RNA strand (i.e. the one directly opposite the non-nucleotide-linker) is sensitive to hydrolytic cleavage catalyzed by ethylenediamine hydrochloride. Furthermore, the regions of the RNA comprising hydrogen bonded domains are resistant to cleavage compared with single-stranded RNA alone. Several catalytic moieties capable of supporting acid/base hydrolysis were coupled to the non-nucleotide-based linker via simple aqueous coupling chemistries. When tethered to the MPO in this manner these moieties are shown to catalyze site-specific cleavage on the RNA target without any additional catalyst.     

Novel immunoconjugates comprised of streptonigrin and 17-amino-geldanamycin attached via a dipeptide-p-aminobenzyl-amine linker system

Cytotoxic agents streptonigrin and 17-amino-geldanamycin were linked to monoclonal antibodies (mAbs), forming antibody–drug conjugates (ADCs) for antigen-mediated targeting to cancer cells. The drugs were conjugated with a linker construct that is labile to lysosomal proteases and incorporates a valine-alanine-p-aminobenzyl (PAB)-amino linkage for direct attachment to the electron-deficient amine functional groups present in both drugs. The resulting ADCs release drug following internalization into antigen-positive cancer cells. The drug linkers were conjugated to mAbs cAC10 (anti-CD30) and h1F6 (anti-CD70) via alkylation of reduced interchain disulfides to give ADCs loaded with 4 drugs/mAb. The streptonigrin ADCs were potent and immunologically specific on a panel of cancer cell lines in vitro and in a Hodgkin lymphoma xenograft model. We conclude that streptonigrin ADCs are candidates for further research, and that the novel linker system used to make them is well-suited for the conjugation of cytotoxic agents containing electron-deficient amine functional groups.     

Contribution of linker stability to the activities of anticancer immunoconjugates

The linker component of antibody-drug conjugates (ADC) is a key feature in developing optimized therapeutic agents that are highly active at well tolerated doses. For maximal intratumoral drug delivery, linkers are required that are highly stable in the systemic circulation, yet allow for efficient drug release at the target site. In this respect, amide bond-based technologies constitute a technological advancement, since the linker half-lives in circulation ( t 1/2 approximately 7 days) are much longer than earlier generation linkers that break down within 1-2 days. The amide linkers, some of which contain peptides, are appended to the mAb carriers through thioether/maleimide adducts. Here, we describe that use of a bromoacetamidecaproyl (bac) in place of the maleimidocaproyl (mc) increases the plasma stability of resulting thioether ADCs. One such ADC, 1F6-C4v2-bac-MMAF, which is directed against the CD70 antigen on lymphomas and renal cell carcinoma, was prepared containing a bac thioether spacer between the drug (MMAF) and the mAb carrier (1F6-C4v2). There was no measurable systemic drug release from this ADC for 2 weeks postadministration in mice. In order to assess the impact of improving linker stability beyond mc containing ADCs, a series of mc and bac-linked 1F6-MMAF conjugates were compared for tolerability, intratumoral drug delivery, and therapeutic efficacy in nude mice with renal cell carcinoma xenografts. There were no statistically significant efficacy differences between sets of mc and bac containing ADCs, although the bac linker technology led to 25% higher intratumoral drug exposure over a 7 day period compared to the corresponding mc linker. The mechanism of drug release from maleimide-adducts likely involves a retro-Michael reaction that takes place in plasma, based on in vitro studies demonstrating that some of the released drug-maleimide derivative became covalently bound to cysteine-34 of serum albumin. In summary, the data indicate that new linkers can be obtained with improved in vivo stability by replacing the maleimide with an acetamide, but the resulting ADCs had similar tolerability and activity profiles.     

An antimelanoma immunotoxin containing recombinant human tumor necrosis factor: tissue disposition,pharmacokinetic, and therapeutic studies in xenograft models

The ability of monoclonal antibody conjugates to re-direct plant or bacterial toxins, chemotherapeutic agents and radionuclides to selected target cells has been well-documented. Recombinant human tumor necrosis factor (TNF) is a macrophage-derived, non-glycosylated (17 kDa) peptide with a broad range of biological and immunological effects including antiviral activity, cytotoxic and cytostatic effects. A conjugate of the antimelanoma antibody ZME-018 and TNF in previous studies has shown melanoma-selective cytotoxic effects in vitro. Pharmacokinetic studies of the ZME-TNF immunotoxin showed that the agent cleared from plasma biphasically with -and -phase half-lives similar to that of ZME itself (72 min and 36 h compared to 84 min and 41 h respectively). In contrast, TNF itself was cleared rapidly from plasma with a terminalphase half-life of only 2.7 h. The clearance rate of ZME-TNF from plasma (Cl_p) was almost tenfold more rapid than for ZME (1.1 versus 0.16 ml/kg x min) but was threefold slower than the clearance for TNF itself (3.4 ml/kg x min). Tissue distribution studies in nude mice bearing human melanoma xenografts showed similar tumor localization of the immunotoxin compared to the free antibody and slightly higher concentrations in liver and kidney compared to ZME itself. Treatment of nude mice bearing well-developed A375 tumors with the immunotoxin resulted in a statistically significant (P<0.002) suppression in tumor growth rate (fivefold increase) compared to saline-treated controls, which increased 20-fold over the same period. These studies demonstrate the feasibility of this approach and suggest that TNF may represent a non-antigenic alternative to immunotoxins containing plant and bacterial toxins.Research conducted, in part, by the Clayton Foundation for Research     

PEG-doxorubicin conjugates: influence of polymer structure on drug release, in vitro cytotoxicity, biodistribution, and antitumor activity

Polymer-drug conjugates (polymer therapeutics) are finding increasing use as novel anticancer agents. Here a series of poly(ethylene glycol) PEG-doxorubicin (Dox) conjugates were synthesized using polymers of linear or branched architecture (molecular weight 5000-20000 g/mol) and with different peptidyl linkers (GFLG, GLFG, GLG, GGRR, and RGLG). The resultant conjugates had a drug loading of 2.7-8.0 wt % Dox and contained <2.0% free drug (% total drug). All conjugates containing a GFLG linker showed approximately 30% release of Dox at 5 h irrespective of PEG molecular weight or architecture. The GLFG linker was degraded more quickly (approximately 57% Dox release at 5 h), and the other linkers more slowly (<16% release at 5 h), by lysosomal enzymes in vitro. In vitro there was no clear relationship between cytotoxicity toward B16F10 cells and the observed Dox release rate. All PEG conjugates were more than 10-fold less toxic (IC50 values > 2 microg/mL) than free Dox (IC50 value = 0.24 microg/mL). Biodistribution in mice bearing sc B16F10 tumors was assessed after administration of PEGs (5000, 10000, or 20000 g/mol) radioiodinated using the Bolton and Hunter reagent or PEG-Dox conjugates by HPLC. The 125I-labeled PEGs showed a clear relationship between Mw and blood clearance and tumor accumulation. The highest Mw PEG had the longest plasma residence time and consequently the greatest tumor targeting. The PEG-Dox conjugates showed a markedly prolonged plasma clearance and greater tumor targeting compared to free Dox, but there was no clear molecular weight-dependence on biodistribution. This was consistent with the observation that the PEG-Dox conjugates formed micelles in aqueous solution comprising 2-20 PEG-Dox molecules depending on polymer Mw and architecture. Although PEG-Dox showed greater tumor targeting than free Dox, PEG conjugation led to significantly lower anthracycline levels in heart. Preliminary experiments to assess antitumor activity against sc B16F10 in vivo showed the PEG5000linear (L)-GFLG-Dox and PEG10000branched (B)-GLFG-Dox (both 5 mg/kg Dox-equiv) to be the most active with T/C values of 146 and 143%, respectively. Free Dox did not show significant activity in this model (T/C = 121%). Dose escalation of PEG5000(L)-GFLG-Dox to 10 mg/kg Dox-equiv prolonged further animal survival (T/C = 161%). Using the Dox-sensitive model ip L1210 (where Dox displayed a T/C = 150% after single ip dose), the PEG5000(L)-GFLG-Dox displayed a maximum T/C of 141% (10 mg/kg Dox-equiv) using a once a day (x3) schedule. Further studies are warranted with PEG5000(L)-GFLG-Dox to determine its spectrum of antitumor activity and also the optimum dosing schedule before clinical testing.     

Strategy for selecting and characterizing linker peptides for CBM9-tagged fusion proteins expressed in Escherichia coli

The influence of linker design on fusion protein production and performance was evaluated when a family 9 carbohydrate-binding module (CBM9) serves as the affinity tag for recombinant proteins expressed in Escherichia coli. Two bioinformatic strategies for linker design were applied: the first identifies naturally occurring linkers within the proteome of the host organism, the second involves screening peptidases and their known specificities using the bioinformatics software MEROPS to design an artificial linker resistant to proteolysis within the host. Linkers designed using these strategies were compared against traditional poly-glycine linkers. Although widely used, glycine-rich linkers were found by tandem MS data to be susceptible to hydrolysis by E. coli peptidases. The natural (PT)(x)P and MEROPS-designed S(3)N(10) linkers were significantly more stable, indicating both strategies provide a useful approach to linker design. Factor X(a) processing of the fusion proteins depended strongly on linker chemistry, with poly(G) and S(3)N(10) linkers showing the fastest cleavage rates. Luminescence resonance energy transfer studies, used to measure average distance of separation between GFP and Tb(III) bound to a strong calcium-binding site of CBM9, revealed that, for a given linker chemistry, the separation distance increases with increasing linker length. This increase was particularly large for poly(G) linkers, suggesting that this linker chemistry adopts a hydrated, extended configuration that makes it particularly susceptible to proteolysis. Differential scanning calorimetry studies on the PT linker series showed that fusion of CBM9 to GFP did not alter the T(m) of GFP but did result in a destabilization, as seen by both a decrease in T(m) and DeltaH(cal), of CBM9. The degree of destabilization increased with decreasing length of the (PT)(x)P linker such that DeltaT(m) = -8.4 degrees C for the single P linker.     

Single-cell fluorescence resonance energy transfer analysis demonstrates that caspase activation during apoptosis is a rapid process. Role of caspase-3

Activation of effector caspases is considered to be the final step in many apoptosis pathways. We transfected HeLa cells with a recombinant caspase substrate composed of cyan and yellow fluorescent protein and a linker peptide containing the caspase cleavage sequence DEVD, and we examined the cleavage kinetics at the single-cell level by fluorescence resonance energy transfer (FRET) analysis. Caspase activation in response to tumor necrosis factor-alpha, staurosporine, or etoposide resulted in cleavage of the linker peptide and subsequent disruption of the FRET signal. The time to caspase activation varied among individual cells, depending on the type of treatment and concentration used. However, once initiated, disruption of the FRET signal was always rapid (相似文献   

Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates

The reactive thiol in cysteine is used for coupling maleimide linkers in the generation of antibody conjugates. To assess the impact of the conjugation site, we engineered cysteines into a therapeutic HER2/neu antibody at three sites differing in solvent accessibility and local charge. The highly solvent-accessible site rapidly lost conjugated thiol-reactive linkers in plasma owing to maleimide exchange with reactive thiols in albumin, free cysteine or glutathione. In contrast, a partially accessible site with a positively charged environment promoted hydrolysis of the succinimide ring in the linker, thereby preventing this exchange reaction. The site with partial solvent-accessibility and neutral charge displayed both properties. In a mouse mammary tumor model, the stability and therapeutic activity of the antibody conjugate were affected positively by succinimide ring hydrolysis and negatively by maleimide exchange with thiol-reactive constituents in plasma. Thus, the chemical and structural dynamics of the conjugation site can influence antibody conjugate performance by modulating the stability of the antibody-linker interface.     

Characterization of several kinds of dimer minizyme: simultaneous cleavage at two sites in HIV-1 tat mRNA by dimer minizymes.

A minizyme is a hammerhead ribozyme with short oligonucleotide linkers instead of stem-loop II. In a previous study we demonstrated that a minizyme with high activity forms a dimeric structure with a common stem II. Because of their dimeric structure, minizymes are potentially capable of cleaving a substrate at two different sites simultaneously. In order to examine the properties of different kinds of minizyme, we constructed a number of minizymes with short oligonucleotide linkers (2-5 bases) instead of stem-loop II and examined their cleavage activities against HIV-1 tat mRNA. Analyses of melting curves, as well as Arrhenius plots, revealed that, in general, the longer the oligonucleotide linkers, the more stable and more active were the dimer minizymes. All minizymes examined cleaved the target substrate at two sites simultaneously. The activity of the dimer minizyme with a 5 nt linker was higher than that of the parental hammerhead ribozyme because the latter full-sized ribozyme was able to cleave at one site only.