Crystal structure and thermodynamic analysis of diagnostic mAb 106.3 complexed with BNP 5‐13 (C10A)

B‐type natriuretic peptide (BNP) is a naturally secreted regulatory hormone that influences blood pressure and vascular water retention in human physiology. The plasma BNP concentration is a clinically recognized biomarker for various cardiovascular diseases. Quantitative detection of BNP can be achieved in immunoassays using the high‐affinity monoclonal IgG1 antibody 106.3, which binds an epitope spanning residues 5‐13 of the mature bioactive peptide. To understand the structural basis of this molecular recognition, we crystallized the Fab fragment complexed with the peptide epitope and determined the three‐dimensional structure by X‐ray diffraction to 2.1 Å resolution. The structure reveals the detailed interactions that five of the complementarity‐determining regions make with the partially folded peptide. Thermodynamic measurements using fluorescence spectroscopy suggest that the interaction is enthalpy driven, with an overall change in free energy of binding, ΔG = ?54 kJ/mol, at room temperature. The parameters are interpreted on the basis of the structural information. The kinetics of binding suggest a diffusion‐limited mechanism, whereby the peptide easily adopts a bound conformation upon interaction with the antibody. Moreover, comparative analysis with alanine‐scanning results of the epitope explains the basis of selectivity for BNP over other related natriuretic peptides. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

A study to assess the cross‐reactivity of cellulose membrane‐bound peptides with detection systems: an analysis at the amino acid level

The growing demand for binding assays to study protein–protein interaction can be addressed by peptide array‐based methods. The SPOT technique is a widespread peptide‐array technology, which is able to distinguish semi‐quantitatively the binding affinities of peptides to defined protein targets within one array. The quality of an assay system used for probing peptide arrays depends on the well‐balanced combination of screening and read‐out methods. The former address the steady‐state of analyte capture, whereas the latter provide the means to detect captured analyte. In all cases, however, false‐positive results can occur when challenging a peptide array with analyte or detecting captured analyte with label conjugates. Little is known about the cross‐reactivity of peptides with the detection agents. Here, we describe at the amino acid level the potential of (i) 5‐(and 6)‐carboxytetramethylrhodamine (5(6)‐TAMRA), (ii) fluoresceinisothiocyanate in form of the peptide‐bound fluorescein‐substituted thiourea derivative (FITC), and (iii) biotin/streptavidin‐POD to cross‐react with individual amino acids in a peptide sequence. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Optimized signal peptides for the development of high expressing CHO cell lines

Recombinant biotherapeutic proteins such as monoclonal antibodies are mostly produced in Chinese hamster ovary (CHO) cells and pharmaceutical companies are interested in an appropriate platform technology for the development of large‐scale production processes. A major aim of our study was therefore to improve the secretion efficiency of a recombinant biotherapeutic antibody by optimizing signal peptides. Reporter molecules such as gaussia and vargula luciferase or secreted alkaline phosphatase are frequently used to this end. In striking contrast, we used a biotherapeutic antibody that was fused to 16 different signal peptides during our study. In this way, the secretion efficiency of the recombinant antibody has been analyzed by transient expression experiments in CHO cell lines. Compared to the control signal peptide, it was not possible to achieve higher efficiencies with signal peptides derived from a variety of species or even natural immunoglobulin G signal peptides. The best results were obtained with natural signal peptides derived from human albumin and human azurocidin. These results were confirmed by fed‐batch experiments with stably transfected cell pools, in which cell‐specific productivities up to 90 pg cell^?1 day^?1 and product concentrations up to 4 g L^?1 could be determined using the albumin signal peptide. Finally, the applicability of the identified signal peptides for both different antibodies and non‐antibody products was demonstrated by transient expression experiments. In conclusion, it was found that signal peptides derived from human albumin and human azurocidin are most appropriate to generate cell lines with clearly improved production rates suitable for commercial purposes in a product‐independent manner. Biotechnol. Bioeng. 2013; 110: 1164–1173. © 2012 Wiley Periodicals, Inc.     

Identification of peptide‐binding sites within BSA using rapid,laser‐induced covalent cross‐linking combined with high‐performance mass spectrometry

We are developing a rapid, time‐resolved method using laser‐activated cross‐linking to capture protein‐peptide interactions as a means to interrogate the interaction of serum proteins as delivery systems for peptides and other molecules. A model system was established to investigate the interactions between bovine serum albumin (BSA) and 2 peptides, the tridecapeptide budding‐yeast mating pheromone (α‐factor) and the decapeptide human gonadotropin‐releasing hormone (GnRH). Cross‐linking of α‐factor, using a biotinylated, photoactivatable p‐benzoyl‐L‐phenylalanine (Bpa)–modified analog, was energy‐dependent and achieved within seconds of laser irradiation. Protein blotting with an avidin probe was used to detect biotinylated species in the BSA‐peptide complex. The cross‐linked complex was trypsinized and then interrogated with nano‐LC–MS/MS to identify the peptide cross‐links. Cross‐linking was greatly facilitated by Bpa in the peptide, but some cross‐linking occurred at higher laser powers and high concentrations of a non‐Bpa–modified α‐factor. This was supported by experiments using GnRH, a peptide with sequence homology to α‐factor, which was likewise found to be cross‐linked to BSA by laser irradiation. Analysis of peptides in the mass spectra showed that the binding site for both α‐factor and GnRH was in the BSA pocket defined previously as the site for fatty acid binding. This model system validates the use of laser‐activation to facilitate cross‐linking of Bpa‐containing molecules to proteins. The rapid cross‐linking procedure and high performance of MS/MS to identify cross‐links provides a method to interrogate protein‐peptide interactions in a living cell in a time‐resolved manner.     

Comparison of marmoset and human FSH using synthetic peptides of the β‐subunit L2 loop region and anti‐peptide antibodies

Follicle stimulating hormone (FSH) is a glycoprotein hormone required for female and male gametogenesis in vertebrates. Common marmoset (Callithrix jacchus) is a New World primate monkey, used as animal model in biomedical research. Observations like, requirement of extremely high dose of human FSH in marmosets for superovulation compared to other primates and generation of antibodies in marmoset against human FSH after repeated superovulation cycles, point towards the possibility that FSH–FSH receptor (FSHR) interaction in marmosets might be different than in the humans. In this study we attempted to understand some of these structural differences using FSH peptides and anti‐peptide antibody approach. Based on sequence alignment, in silico modeling and docking studies, L2 loop of FSH β‐subunit (L2β) was found to be different between marmoset and human. Hence, peptides corresponding to region 32–50 of marmoset and human L2β loop were synthesized, purified and characterized. The peptides displayed dissimilarity in terms of molecular mass, predicted isoelectric point, predicted charge and in the ability to inhibit hormone–receptor interaction. Polyclonal antibodies generated against both the peptides were found to exhibit specific binding for the corresponding peptide and parent FSH in ELISA and Western blotting respectively and exhibited negligible reactivity to cross‐species peptide and FSH in ELISA. The anti‐peptide antibody against marmoset FSH was also able to detect native FSH in marmoset plasma samples and pituitary sections. In summary, the L2β loop of marmoset and human FSH has distinct receptor interaction ability and immunoreactivity indicating possibility of subtle conformational and biochemical differences between the two regions which may affect the FSH–FSHR interaction in these two primates. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Comparison of patient‐derived high and low phosphatidylserine‐exposing colorectal carcinoma cells in their interaction with anti‐cancer peptides

Current cancer treatment is frequently compromised by severe adverse effects on healthy cells and tissues as well as by the increasing burden of (multi‐)drug resistances. Some representatives of small, amphipathic peptides known as host defense peptides possess the potential to overcome these limitations and to evolve as future anti‐cancer therapeutics. Peptide NK‐2, derived from porcine NK‐lysin, was originally discovered due to its broad‐spectrum antimicrobial activities. Today, also potent anti‐cancer activity is proven and accompanied by low toxicity towards normal human cells. The molecular basis underlying this target selectivity remains rather elusive. Nevertheless, it is presumptive that preferential peptide interactions with surface factors non‐abundant on healthy human cells play a key role. Here, we investigated the cytotoxicity of peptide NK‐2 and structurally improved anti‐cancer variants thereof against two patient‐derived colorectal cancer cell lines, exposing high and low levels of phosphatidylserine on their cell surfaces, respectively. Concluding from a range of in vitro tests involving cellular as well as lipid vesicle‐based methods, it is proposed that the magnitude of the accessible membrane surface charge is not a primarily decisive factor for selective peptide interactions. Instead, it is suggested that the level of membrane surface‐exposed phosphatidylserine is of crucial importance for the activity of peptide NK‐2 and enhanced variants thereof in terms of their cancer cell selectivity, the overall efficacy, as well as the underlying mode of action and kinetics. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and properties of cyclic gomesin and analogues

Gomesin (Gm) was the first antimicrobial peptide (AMP) isolated from the hemocytes of a spider, the Brazilian mygalomorph Acanthoscurria gomesiana. We have been studying the properties of this interesting AMP, which also displays anticancer, antimalarial, anticryptococcal and anti‐Leishmania activities. In the present study, the total syntheses of backbone‐cyclized analogues of Gm (two disulfide bonds), [Cys(Acm)^2,15]‐Gm (one disulfide bond) and [Thr^2,6,11,15,d ‐Pro⁹]‐Gm (no disulfide bonds) were accomplished, and the impact of cyclization on their properties was examined. The consequence of simultaneous deletion of pGlu¹ and Arg¹⁶‐Glu‐Arg¹⁸‐NH₂ on Gm antimicrobial activity and structure was also analyzed. The results obtained showed that the synthetic route that includes peptide backbone cyclization on resin was advantageous and that a combination of 20% DMSO/NMP, EDC/HOBt, 60 °C and conventional heating appears to be particularly suitable for backbone cyclization of bioactive peptides. The biological properties of the Gm analogues clearly revealed that the N‐terminal amino acid pGlu¹ and the amidated C‐terminal tripeptide Arg¹⁶‐Glu‐Arg¹⁸‐NH₂ play a major role in the interaction of Gm with the target membranes. Moreover, backbone cyclization practically did not affect the stability of the peptides in human serum; it also did not affect or enhanced hemolytic activity, but induced selectivity and, in some cases, discrete enhancements of antimicrobial activity and salt tolerance. Because of its high therapeutic index, easy synthesis and lower cost, the [Thr^2,6,11,15,d ‐Pro⁹]‐Gm analogue remains the best active Gm‐derived AMP developed so far; nevertheless, its elevated instability in human serum may limit its therapeutic potential. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Cross‐reactivity of a human IgG1 anticitrullinated fibrinogen monoclonal antibody to a citrullinated profilaggrin peptide

Rheumatoid arthritis (RA) is the most common autoimmune rheumatic disease. It is characterized by persistent joint inflammation, resulting in loss of joint function, morbidity and premature mortality. The presence of antibodies against citrullinated proteins is a characteristic feature of RA and up to 70% of RA patients are anticitrullinated protein antibody (ACPA) positive. ACPA responses have been widely studied and are suggested to be heterogeneous, favoring antibody cross‐reactivity to citrullinated proteins. In this study, we examined factors that may influence cross‐reactivity between a commercial human anticitrullinated fibrinogen monoclonal antibody and a citrullinated peptide. Using a citrullinated profilaggrin sequence (HQCHQEST‐ Cit‐GRSRGRCGRSGS) as template, cyclic and linear truncated peptide versions were tested for reactivity to the monoclonal antibody. Factors such as structure, peptide length and flanking amino acids were found to have a notable impact on antibody cross‐reactivity. The results achieved contribute to the understanding of the interactions between citrullinated peptides and ACPA, which may aid in the development of improved diagnostics of ACPA.     

Epitope mapping of a monoclonal antibody directed against the α‐subunit of phosphofructokinase‐1 from Saccharomyces cerevisiae by screening phage display libraries

Phosphofructokinase‐1 from Saccharomyces cerevisiae is composed of two types of subunits, α and β. Subunit‐specific monoclonal antibodies were raised to elucidate structural and functional properties of both subunits. One monoclonal antibody, α‐F3, binds to an epitope either at the C‐terminal or at the N‐terminal part of the α‐polypeptide chain. By screening a heptapeptide library with this monoclonal antibody, a set of heptapeptides was selected, which contained the consensus sequences D–A–F and D–S–F. Two heptapeptides with these motifs were synthesized in order assess their capacity to inhibit the binding of antibody α‐F3 to native phosphofructokinase‐1. The peptide G–I–K–D–A–F–L inhibited the binding more strongly (IC₅₀ = 1.5 µM) than the peptide A–P–W–H–D–S–F (IC₅₀ = 33.3 µM). Sequence matching revealed the presence of the D–A–F motif in the polypeptide chain of phosphofructokinase‐1 at amino acid position 172–174. As a control, the nonapeptide A–P–T–S–K–D–A–F–L which corresponds to the sequence of the putative epitope was tested in the inhibition assay. In view of the high inhibitory capacity (IC₅₀ = 0.3 µM) it was concluded that this nonapeptide represents the continuous epitope of phosphofructokinase‐1 that is recognized by antibody α‐F3. Copyright © 1999 John Wiley & Sons, Ltd.     

Characterization of antilytic peptide antibody: application for the detection of lytic‐based hybrid peptide in serum samples

We previously reported that a novel targeted drug termed hybrid epidermal growth factor receptor (EGFR)‐lytic peptide, made by chemical conjugation of targeted binding peptide and cell‐killing, lytic‐peptide components, has selective cytotoxic activity that allows it to discriminate between normal and cancer cells. In addition, in vivo analysis revealed that this hybrid peptide displays significant antitumor activity in a xenograft model of human breast and pancreatic cancer in mice. Here, we characterized antilytic peptide antibody, which was raised from rabbit serum using the antigen of lytic peptide conjugated with keyhole limpet hemocyanin. It was found that antilytic peptide antibody is specific to the lytic peptide as assessed by both ELISA and surface plasmon resonance analysis and can also bind to EGFR‐lytic peptide. Epitope mapping analysis using Biacore showed that two successive lysine regions in the lytic‐peptide sequence are significant for recognition by this antibody. In addition, it was shown that this antibody can detect lytic‐based hybrid peptide in serum samples from mouse blood and also in cultured breast cancer MDA‐MB‐231 cell samples by immunocytochemical staining experiments. It was found that the maximum concentrations of this peptide in serum were reached within 15–30 min of i.v. administration of EGFR‐lytic peptide to mice. These results indicate that this antibody will be a useful tool for the detection of lytic‐based peptides to investigate their in vivo stability and pharmacokinetics. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Conformational dynamics of a short antigenic peptide in its free and antibody bound forms gives insight into the role of β‐turns in peptide immunogenicity

Earlier immunological experiments with a synthetic 36‐residue peptide (75‐110) from Influenza hemagglutinin have been shown to elicit anti‐peptide antibodies (Ab) which could cross‐react with the parent protein. In this article, we have studied the conformational features of a short antigenic (Ag) peptide (⁹⁸YPYDVPDYASLRS¹¹⁰) from Influenza hemagglutinin in its free and antibody (Ab) bound forms with molecular dynamics simulations using GROMACS package and OPLS‐AA/L all‐atom force field at two different temperatures (293 K and 310 K). Multiple simulations for the free Ag peptide show sampling of ordered conformations and suggest different conformational preferences of the peptide at the two temperatures. The free Ag samples a conformation crucial for Ab binding (β‐turn formed by “DYAS” sequence) with greater preference at 310 K while, it samples a native‐like conformation with relatively greater propensity at 293 K. The sequence “DYAS” samples β‐turn conformation with greater propensity at 310 K as part of the hemagglutinin protein also. The bound Ag too samples the β‐turn involving “DYAS” sequence and in addition it also samples a β‐turn formed by the sequence “YPYD” at its N‐terminus, which seems to be induced upon binding to the Ab. Further, the bound Ag displays conformational flexibility at both 293 K and 310 K, particularly at terminal residues. The implications of these results for peptide immunogenicity and Ag–Ab recognition are discussed. Proteins 2015; 83:1352–1367. © 2015 Wiley Periodicals, Inc.     

Variant antibody identification by peptide mapping

During development of CGP56901, a monoclonal antibody (MAb) specific for a unique epitope on human IgE, the protein A‐purified IgG from one of the candidate production cell lines, showed an additional minor heavy chain (H‐chain) band with a molecular weight slightly lower than that of the principal H‐chain band on SDS‐PAGE. The N‐terminal amino acid sequence of this minor H‐chain species indicated that at least the first 30 amino acids were identical to those of the antibody light‐chain (L‐chain) variable domain. More detailed studies using peptide mapping and amino acid sequencing analysis confirmed a crossover event between the V genes of the antibody. The position is between Arg¹⁰⁸ of the L chain and Ala¹²⁴ of the H chain. This crossover resulted in a variant H chain, which had 16 fewer amino acid residues than the normal CGP56901 H chain. These results show that peptide mapping is a useful “first‐line” analytical tool in the characterization of the quality of the monoclonal antibody. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 485–488, 1999.     

Chemically synthesized peptide libraries as a new source of BBB shuttles. Use of mass spectrometry for peptide identification

The blood–brain barrier (BBB) is a biological barrier that protects the brain from neurotoxic agents and regulates the influx and efflux of molecules required for its correct function. This stringent regulation hampers the passage of brain parenchyma‐targeting drugs across the BBB. BBB shuttles have been proposed as a way to overcome this hurdle because these peptides can not only cross the BBB but also carry molecules which would otherwise be unable to cross the barrier unaided. Here we developed a new high‐throughput screening methodology to identify new peptide BBB shuttles in a broadly unexplored chemical space. By introducing d‐ amino acids, this approach screens only protease‐resistant peptides. This methodology combines combinatorial chemistry for peptide library synthesis, in vitro models mimicking the BBB for library evaluation and state‐of‐the‐art mass spectrometry techniques to identify those peptides able to cross the in vitro assays. BBB shuttle synthesis was performed by the mix‐and‐split technique to generate a library based on the following: Ac‐d‐ Arg‐XXXXX‐NH₂, where X were: d‐ Ala (a), d‐ Arg (r), d‐ Ile (i), d‐ Glu (e), d‐ Ser (s), d‐ Trp (w) or d‐ Pro (p). The assays used comprised the in vitro cell‐based BBB assay (mimicking both active and passive transport) and the PAMPA (mimicking only passive diffusion). The identification of candidates was determined using a two‐step mass spectrometry approach combining LTQ‐Orbitrap and Q‐trap mass spectrometers. Identified sequences were postulated to cross the BBB models. We hypothesized that some sequences cross the BBB through passive diffusion mechanisms and others through other mechanisms, including paracellular flux and active transport. These results provide a new set of BBB shuttle peptide families. Furthermore, the methodology described is proposed as a consistent approach to search for protease‐resistant therapeutic peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Epitope‐specificity of recombinant antibodies reveals promiscuous peptide‐binding properties

Protein–peptide interactions are a common occurrence and essential for numerous cellular processes, and frequently explored in broad applications within biology, medicine, and proteomics. Therefore, understanding the molecular mechanism(s) of protein–peptide recognition, specificity, and binding interactions will be essential. In this study, we report the first detailed analysis of antibody–peptide interaction characteristics, by combining large‐scale experimental peptide binding data with the structural analysis of eight human recombinant antibodies and numerous peptides, targeting tryptic mammalian and eukaryote proteomes. The results consistently revealed that promiscuous peptide‐binding interactions, that is, both specific and degenerate binding, were exhibited by all antibodies, and the discovery was corroborated by orthogonal data, indicating that this might be a general phenomenon for low‐affinity antibody–peptide interactions. The molecular mechanism for the degenerate peptide‐binding specificity appeared to be executed through the use of 2–3 semi‐conserved anchor residues in the C‐terminal part of the peptides, in analogue to the mechanism utilized by the major histocompatibility complex–peptide complexes. In the long‐term, this knowledge will be instrumental for advancing our fundamental understanding of protein–peptide interactions, as well as for designing, generating, and applying peptide specific antibodies, or peptide‐binding proteins in general, in various biotechnical and medical applications.     

Epitope analysis of the multiphosphorylated peptide αs1‐casein(59–79)

The multiphosphorylated tryptic peptide α_s1‐casein(59–79) has been shown to be antigenic with anti‐casein antibodies. In an approach to determine the amino acyl residues critical for antibody binding we undertook an epitope analysis of the peptide using overlapping synthetic peptides. With α_s1‐casein(59–79) as the adsorbed antigen in a competitive ELISA only two of five overlapping synthetic peptides at 1 mM significantly inhibited binding of the anti‐casein antibodies. Peptides Glu‐Ser(P)‐Ile‐Ser(P)‐Ser(P)‐Ser(P)‐Glu‐Glu and Ile‐Val‐Pro‐Asn‐Ser(P)‐Val‐Glu‐Glu inhibited antibody binding by 20.0±3.6% and 60.3±7.9%, respectively. The epitope of Glu⁶³‐Ser(P)‐Ile‐Ser(P)‐Ser(P)‐Ser(P)‐Glu‐Glu⁷⁰ was further localised to the phosphoseryl cluster as the peptide Ser(P)‐Ser(P)‐Ser(P) significantly inhibited binding of the anti‐casein antibodies to α_s1‐casein(59–79) by 29.5±7.4%. Substitution of Ser(P)⁷⁵ with Ser⁷⁵ in the second inhibitory peptide Ile‐Val‐Pro‐Asn‐Ser(P)⁷⁵‐Val‐Glu‐Glu also abolished inhibition of antibody binding to α_s1‐casein (59–79) demonstrating that Ser(P)⁷⁵ is also a critical residue for recognition by the antibodies. These data show that the phosphorylated residues in the cluster sequence ‐Ser(P)⁶⁶‐Ser(P)‐Ser(P)⁶⁸ and in the sequence ‐Pro⁷³‐Asn‐Ser(P)‐Val‐Glu⁷⁷‐ are critical for antibody binding to α_s1‐casein(59–79) and further demonstrate that a highly phosphorylated segment of a protein can be antigenic. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Evidence for conformationally different states of interleukin‐10: binding of a neutralizing antibody enhances accessibility of a hidden epitope

We present the mapping of two anti‐human interleukin‐10 (hIL‐10) antibodies (CB/RS/2 and CB/RS/11) which have been described as binding their antigen cooperatively. The epitopes were identified using hIL‐10‐derived overlapping peptide scans prepared by spot synthesis. To identify residues essential for binding within the two epitopes, each position was replaced by all other L ‐amino acids. The epitope‐derived peptides were further characterized with respect to antibody affinity and their inhibition of the antibody–hIL‐10 interaction. One antibody (CB/RS/11) binds to residues which are completely buried in the X‐ray structure of IL‐10. Accessibility of this hidden epitope is enhanced upon binding of the antibody CB/RS/2, which recognizes a discontinuous epitope located nearby. The recognition of the hidden CB/RS/11 epitope, as well as the cooperative binding behaviour of the two antibodies, provides evidence that IL‐10 can adopt a conformational state other than that observed in the crystal structure. Copyright © 1999 John Wiley & Sons, Ltd.     

Molecular design of Stat3‐derived peptide selectivity between BET proteins Brd2 and Brd4 in ovarian cancer

Stat3 signaling has been recognized as a potential therapeutic target of human ovarian cancer. The signaling is transducted through the peptide‐medicated interaction of Stat3 with BET family members Brd2 and Brd4 –– 2 highly homologous proteins involved in differential downstream pathways. Here, we reported a successful design of peptide selectivity between the Brd2 and Brd4. The design resulted in 3 linear peptides SMSLQCX YLGVA, QSKVLTX SYWGA, and RQCNLGX LYMNY with high or moderate selectivity for Brd2 over Brd4 (S = 3.3‐fold, 6.8‐fold, and 4.2‐fold, respectively) as compared with the native Stat3 peptide ²⁸¹HNLLRIX QFLQS²⁹² (S = 2.5‐fold). Structural analysis revealed that peptide N‐terminus and hydrogen bonds play important roles in the peptide interaction stability and specificity with Brd2 and Brd4. This study would help to establish an integrated in silico‐in vitro method for rational molecular design of peptide ligand selectivity between homologous protein receptors.     

Cystine‐knot peptides targeting cancer‐relevant human cytotoxic T lymphocyte‐associated antigen 4 (CTLA‐4)

Cystine‐knot peptides sharing a common fold but displaying a notably large diversity within the primary structure of flanking loops have shown great potential as scaffolds for the development of therapeutic and diagnostic agents. In this study, we demonstrated that the cystine‐knot peptide MCoTI‐II, a trypsin inhibitor from Momordica cochinchinensis, can be engineered to bind to cytotoxic T lymphocyte‐associated antigen 4 (CTLA‐4), an inhibitory receptor expressed by T lymphocytes, that has emerged as a target for the treatment of metastatic melanoma. Directed evolution was used to convert a cystine‐knot trypsin inhibitor into a CTLA‐4 binder by screening a library of variants using yeast surface display. A set of cystine‐knot peptides possessing dissociation constants in the micromolar range was obtained; the most potent variant was synthesized chemically. Successive conjugation with neutravidin, fusion to antibody Fc domain or the oligomerization domain of C4b binding protein resulted in oligovalent variants that possessed enhanced (up to 400‐fold) dissociation constants in the nanomolar range. Our data indicate that display of multiple knottin peptides on an oligomeric scaffold protein is a valid strategy to improve their functional affinity with ramifications for applications in diagnostics and therapy. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Impact of antibody aggregation on a flowthrough anion‐exchange membrane process

The impact of typical anion‐exchange flowthrough conditions on the IgG mass loading of an anion‐exchange membrane scale‐down unit (Mustang® Q coin) was investigated. High performance size‐exclusion chromatography and multiangle laser light scattering results suggested the presence of a small fraction of IgG aggregates with average radius >100 nm under anion‐exchange flowthrough conditions. The small filtration area presented by the 0.35 mL membrane volume Mustang® Q coin limited the membrane throughput due to fouling from the aggregates at higher antibody loading. Data in this report indicated that a 0.2 μm hybrid polyethersulfone and polyvinylidene fluoride membrane in‐line prefilter with a minimum filtration area of 20 sq cm alleviated the Mustang® Q coin fouling. The combined cake filtration and intermediate blocking model was proposed as the most likely membrane pore blocking mechanism. Increasing the filtration area in the in‐line prefilter resulted in higher IgG mass throughput. Thus, using an appropriately sized in‐line prefilter could provide more robust antibody throughput performance on scale‐down membrane anion‐exchange units. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010