Phagemid Library Research Articles

Generation of Antibody Libraries for Phage Display: Preparation of Helper Phage.

The generation and selection of antibody libraries by phagemid-based phage display requires three components; namely, phagemid library, host bacterial cells, and helper phage. The use of helper phage is necessary for the selection of phagemid libraries by phage display because it provides all genes needed for production of infectious phage particles. Here, we describe the generation of high-titer helper phage preparations suitable for phagemid-based phage display. The approach is based on helper phage VCSM13, which includes a gene for kanamycin resistance and a mutated packaging signal that, in the presence of a phagemid with an unmutated packaging signal, favors the production of infectious phage particles with phagemid phenotype and genotype.

Antibody fragments targeting the extracellular domain of follicular stimulating hormone receptor for contraception in male dogs and cats

The increased LH levels resulting from the absence of negative feedback after castration has been linked to long-term health issues. A need exists for an alternative contraceptive agent that functions without interfering the LH pathways. This study aimed to develop antibody fragments against the follicular-stimulating hormone receptor (anti-FSHr) using phage-display technology and evaluate its effects on Sertoli cell functions. Phage clones against the extracellular domain of dog and cat FSHr selected from an antibody fragment phagemid library were analyzed for binding kinetics by surface plasmon resonance. Sertoli cells were isolated from testes of adult animals (five dogs and five cats). Efficacy test was performed by treating Sertoli cell cultures (SCCs) with anti-FSHr antibody fragments compared with untreated in triplicates. Expressions of androgen binding protein (ABP), inhibin subunit beta B (IHBB) and vascular endothelial growth factor A (VEGFA) mRNA in SCCs were quantified by RT-qPCR. The results demonstrated that the molecular weight of the purified dog and cat anti-FSHr antibody fragment was 25 kDa and 15 kDa, respectively. Based on protein molecular weight, the antibody fragment of dogs and cats was therefore, so-called single-chain variable fragments (scFv) and nanobody (nb), respectively. The binding affinity with dissociation constant (KD) was 2.32 × 10−7 M and 2.83 × 10−9 M for dog and cat anti-FSHr antibody fragments, respectively. The cross-binding kinetic interactions between the dog anti-FSHr scFv and the cat ECD of FSHr could not be fitted to the curves to determine the binding kinetics. However, the cross-binding affinity KD between the cat anti-FSHr nb and the dog ECD FSHr was 1.75 × 10−4 M. The mRNA expression of ABP, IHBB and VEGFA in SCCs was less (P < 0.05) in both dogs (12.26, 4.07 and 5.11 folds, respectively) and cats (39.53, 14.07 and 20.29 folds, respectively) treated with anti-FSHr antibody fragments, indicating the Sertoli cell functions were suppressed. In conclusion, this study demonstrated the establishment of species-specific antibody fragments against FSHr in SCCs for dogs and cats. The fragment proteins illustrate potential to be developed as non-surgical contraceptive agent targeting FSHr in companion animals.

A Detailed Protocol for Constructing a Human Single-Chain Variable Fragment (scFv) Library and Downstream Screening via Phage Display.

The development of monoclonal antibodies (mAbs) represents a significant milestone in both basic research and clinical applications due to their target specificity and versatility in therapeutic and diagnostic applications. The innovative strategy of mAb screening, utilizing phage display, facilitates the in vitro screening of antibodies with high affinity to target antigens. The single-chain variable fragment (scFv) is a subset of mAb derivatives, known for its high binding affinity and smaller size-just one-third of that of human IgG. This report outlines a detailed and comprehensive procedure for constructing a scFv phagemid library derived from human patients, followed by screening via phage display affinity selection. The protocol utilizes 348 primer combinations spanning the entire human antibody repertoire to minimize sequence bias and maintain library diversity during polymerase chain reaction (PCR) for scFv generation, resulting in a library size greater than 1 × 108. Furthermore, we describe a high-throughput phage display screening protocol using enzyme-linked immunosorbent assay (ELISA) to evaluate more than 1200 scFv candidates. The generation of a highly diverse scFv library, coupled with the implementation of a phage display screening methodology, is expected to provide a valuable resource for researchers in pursuit of scFvs with high affinity for target antigens, thus advancing both research and clinical endeavors.

Impaired Testicular Function without Altering Testosterone Concentration Using an Anti-Follicular-Stimulating Hormone Receptor (Anti-FSHr) Single-Chain Variable Fragment (scFv) in Long-Tailed Macaques (Macaca fascicularis).

FSHr antibodies have been shown to inhibit the differentiation of spermatogonia to primary spermatocytes, resulting in infertility without a pathological effect on reproductive organs. The aim of this study was to develop single-chain variable fragments (scFvs) against the follicular-stimulating hormone receptor (anti-FSHr) using phage-display technology and to evaluate the effects of intratesticular administration of the anti-FSHr scFv on testicular function and testosterone production. A phage clone against the extracellular domain of FSHr selected from a scFv phagemid library was analyzed for binding kinetics by surface plasmon resonance. Using ultrasound guidance, three adult macaques (M. fascicularis) were administered with 1 mL of 0.4 mg/mL anti-FSHr scFv (treatment) and 1 mL sterile phosphate buffer solution (control) into the left and right rete testis, respectively. Testicular appearance and volume, ejaculate quality, and serum testosterone levels were recorded on day 0 (before injection) and on days 7, 28, and 56 (after injection). Testicular tissue biopsies were performed on day 7 and day 56 to quantify the mRNA expressions of androgen binding protein (ABP), inhibin subunit beta B (IHBB), and vascular endothelial growth factor A (VEGFA). The results demonstrated that the anti-FSHr scFv molecule was calculated as 27 kDa with a dissociation constant (KD) of 1.03 µM. The volume of the anti-FSHr scFv-injected testicle was reduced on days 28 and 56 compared with day 0 (p < 0.05). Total sperm number was reduced from day 0 (36.4 × 106 cells) to day 56 (1.6 × 106 cells) (p < 0.05). The percentage of sperm motility decreased from day 0 (81.7 ± 1.0%) to day 7 (23.3 ± 1.9%), day 28 (41.7 ± 53.4%), and day 56 (8.3 ± 1.9%) (p < 0.05). Sperm viability on day 0 was 86.8 ± 0.5%, which reduced to 64.2 ± 1.5%, 67.1 ± 2.2%, and 9.3 ± 1.1% on days 7, 28, and 56, respectively (p < 0.05). The expression of ABP and VEGFA on days 7 (14.2- and 3.2-fold) and 56 (5.6- and 5.5-fold) was less in the scFv-treated testicle compared with the controls (p < 0.05). On day 56, the expression of IHBB was less (p < 0.05) in the treated testis (1.3-fold) compared with the controls. Serum testosterone levels were unchanged throughout the study period (p > 0.05). This study characterized the anti-FSHr scFv and demonstrated that treatment with anti-FSHr ameliorates testicular function without altering testosterone levels, offering a potential alternative contraceptive for the long-tailed macaques.

A bispecific antibody AP203 targeting PD-L1 and CD137 exerts potent antitumor activity without toxicity

BackgroundBispecific antibody has garnered considerable attention in the recent years due to its impressive preliminary efficacy in hematological malignancies. For solid tumors, however, the main hindrance is the suppressive tumor microenvironment, which effectively impedes the activation of infiltrating T cells. Herein, we designed a bispecific antibody AP203 with high binding affinity to PD-L1 and CD137 and assessed its safety and anti-tumor efficacy, as well as explored the mechanism of action.MethodsThe optimal antibody binders against PD-L1 and CD137 were screened from the OmniMab phagemid library. The binding affinity of the constructed AP203 were evaluated using enzyme-linked immunosorbent assay (ELISA) and biolayer interferometry (BLI). T-cell stimulatory capacity was assessed using the allogeneic mixed lymphocyte reaction (MLR), antigen-specific recall response, and coculture with PD-L1-expressing cells. In vivo antitumor efficacy was evaluated using two models of tumor-xenografted humanized mice with profiling of tumor infiltrating lymphocytes (TILs). The possible toxicity of AP203 was examined using in vitro cytokine release assay by human PBMCs.ResultsAP203, which simultaneously targeted PD-L1 and costimulatory CD137, elicit superior agonistic effects over parental antibodies alone or in combination in terms of T cell activation, enhanced memory recall responses, and overcoming Treg-mediated immunosuppression (P < 0.05). The agonistic activity of AP203 was further demonstrated PD-L1-dependent by coculturing T cells with PD-L1-expressing cells. In vivo animal studies using immunodeficient or immunocompetent mice both showed a dose-related antitumor efficacy superior to parental antibodies in combination (P < 0.05). Correspondingly, AP203 significantly increased tumor infiltrating CD8 + T cells, while decreased CD4 + T cells, as well as Treg cells (P < 0.05), resulting in a dose-dependent increase in the CD8 + /CD4 + ratio. Moreover, either soluble or immobilized AP203 did not induce the production of inflammatory cytokines by human PBMCs.ConclusionsAP203 exerts potent antitumor activity not only by blocking PD-1/PD-L1 inhibitory signaling, but also by activating CD137 costimulatory signaling in effector T cells that consequently counteracts Treg-mediated immunosuppression. Based on promising preclinical results, AP203 should be a suitable candidate for clinical treatment of solid tumors.

Selection of Affibody Affinity Proteins from Phagemid Libraries.

Herein, we describe a general protocol for the selection of target-binding affinity protein molecules from a phagemid-encoded library. The protocol is based on our experience with phage display selections of non-immunoglobulin affibody affinity proteins but can in principle be applied to perform biopanning experiments from any phage-displayed affinity protein library available in a similar phagemid vector. The procedure begins with an amplification of the library from frozen bacterial glycerol stocks via cultivation and helper phage superinfection, followed by a step-by-step instruction of target protein preparation, selection cycles, and post-selection analyses. The described procedures in this standard protocol are relatively conservative and rely on ordinary reagents and equipment available in most molecular biology laboratories.

Phage-assisted, active site-directed ligand evolution of a potent and selective histone deacetylase 8 inhibitor.

Phage-assisted, active site-directed ligand evolution (PADLE) is a recently developed technique that uses an amber codon-encoded noncanonical amino acid (ncAA) as an anchor to direct phage-displayed peptides to a target for an enhanced ligand identification process. 2-Amino-8-oxodecanoic acid (Aoda) is a ketone-containing ncAA residue in the macrocyclic peptide natural product apicidin that is a pan-inhibitor of Zn2+ -dependent histone deacetylases (HDACs). Its ketone serves as an anchoring point to coordinate the catalytic zinc ion in HDACs. Using a previously evolved N𝜀 -acetyl-lysyl-tRNA synthetase in combination with tRNAPyl , we showed that Aoda was efficiently incorporated into proteins in Escherichia coli by amber suppression. By propagating an amber codon-obligate phagemid library in E. coli encoding Aoda, we generated an Aoda-containing phage-displayed peptide library. Using this library to conduct PADLE against HDAC8 revealed a 7-mer peptide GH8P01F1 with Aoda-flanking amino acid residues that matched existing peptide sequences in identified HDAC8 substrates. Switching Aoda in GH8P01F1 to a more Zn2+ -chelating ncAA S-2-amino-8-hydroxyamino-8-oxooctanoic acid (Asuha) led to an extremely potent compound GH8HA01, which has an HDAC8-inhibition Ki value of 0.67 nM. GH8HA01 and its 5-mer truncation analogue Ac-GH8HA01Δ1Δ7 that has an HDAC8-inhibition Ki value of 0.31 nM are two of the most potent HDAC8 inhibitors that have been developed. Furthermore, both are highly selective against HDAC8 compared with other HDACs tested, demonstrating the great potential of using PADLE to identify highly potent and selective ligands for targets with conserved active sites among homologues.

Selection and Characterization of a Single-Chain Variable Fragment against Porcine Circovirus Type 2 Capsid and Impedimetric Immunosensor Development.

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated disease (PCVAD) that causes huge global economic losses for the swine industry. Effective strategies or rapid detection of PCV2 in pig are essential to control PCVAD. Here, single-chain variable fragments (scFvs) were selected and characterized against the PCV2 capsid using phage display technology. Phage scFv clones were selected from the human scFv phagemid library (Tomlinson I + J) for direct panning against the PCV2 capsid. Eighty-four monoclonal phage scFvs were individually tested for binding to the PCV2 capsid by ELISA. Eight scFv clones showed significant binding to the PCV2 capsid and only three clones (clone nos. 13, 37, and 81) contained both VHCDRs and VLCDRs in the sequence. Clone scFv no. 81 had the highest reactivity to the PCV2 capsid and was constructed in the pET22b (+) expression vector. The recombinant was transformed to Escherichia coli BL21(DE3) for expression and purification. The scFv showed appropriate affinity to the PCV2 capsid by western blot analysis. Kinetics of scFv and the PCV2 capsid were determined using surface plasmon resonance and showed binding affinity in the nanomolar range (KD = 57.2 nM). Our scFv was first applied in the development of an impedimetric immunosensor for PCV2 capsid detection, and results showed that impedance increased with increasing PCV2 capsid expression with limit of detection = 114 nM. Findings demonstrated that our scFv has potential for use as a receptor for biosensor devices.

Using Phage Display to Develop Ubiquitin Variant Modulators for E3 Ligases

Ubiquitin is a small 8.6 kDa protein that is a core component of the ubiquitin-proteasome system. Consequently, it can bind to a diverse array of proteins with high specificity but low affinity. Through phage display, ubiquitin variants (UbVs) can be engineered such that they exhibit improved affinity over wildtype ubiquitin and maintain binding specificity to target proteins. Phage display utilizes a phagemid library, whereby the pIII coat protein of a filamentous M13 bacteriophage (chosen because it is displayed externally on the phage surface) is fused with UbVs. Specific residues of human wildtype ubiquitin are soft and randomized (i.e., there is a bias towards to native wildtype sequence) to generate UbVs so that deleterious changes in protein conformation are avoided while introducing the diversity necessary for promoting novel interactions with the target protein. During the phage display process, these UbVs are expressed and displayed on phage coat proteins and panned against a protein of interest. UbVs that exhibit favorable binding interactions with the target protein are retained, whereas poor binders are washed away and removed from the library pool. The retained UbVs, which are attached to the phage particle containing the UbV's corresponding phagemid, are eluted, amplified, and concentrated so that they can be panned against the same target protein in another round of phage display. Typically, up to five rounds of phage display are performed, during which a strong selection pressure is imposed against UbVs that bind weakly and/or promiscuously so that those with higher affinities are concentrated and enriched. Ultimately, UbVs that demonstrate higher specificity and/or affinity for the target protein than their wildtype counterparts are isolated and can be characterized through further experiments.

Nanobody LAMPoles: Novel tools for dengue virus and Zika virus diagnosis

Abstract Detection of antibodies by ELISA for verifying exposure to dengue virus (DENV) and Zika virus (ZIKV) has been a challenge. Point-of-care (POC) diagnostics with detection of very low concentrations of viral antigens would improve critical disease staging and surveillance to reduce morbidity and mortality. LAMPoles are protein-DNA hybrids where a ‘DNA barcode’ is covalently linked to a single site on a protein. Recombinant camelid nanobodies (Nb), which are variable region of the heavy chain antigen binding domain (VHH), can be tailored to bind almost any molecular target to maximize assay sensitivity and specificity. LAMPole fusions displaying Nb that bind ZIKV and DENV antigens will improve the sensitivity and specificity of virus antigen measurement at the POC. Llama were vaccinated with recombinant DENV envelope or ZIKV NS1 proteins and VHH phagemid libraries constructed and screened for DENV and ZIKV antigen-specific Nb. For DENV 70 full-length Nb were identified, belonging to 26 different CDR3 groups. For ZIKV 63 full-length anti-ZIKV Nb were identified, belonging to 42 different CDR3 groups. Nb screened by surface plasmon resonance spectroscopy with Koff kinetics in the 10−3 sec range were conjugated to chalcone synthase specific DNA elements using psoralen-based photocross-linkers. In a pilot experiment, LAMP targeting the CHS DNA tail of one Nb-LAMPole detected less than 10 fg of Nb-LAMPole protein. Nb-LAMPole recognized DENV-2 E-protein antigen in sandwich ELISA and in DENV-2 infected cells by immunofluorescence. We assert that LAMPole fusions displaying Nb that bind viral antigens may improve the sensitivity and specificity of DENV and ZIKV antigen detection that will facilitate markedly improved POC diagnosis.

Systemic lupus erythematosus: Possible localization of trypsin-like and metalloprotease active centers in the protein sequence of the monoclonal light chain (NGTA2-Me-pro-Tr).

It was previously shown that several monoclonal light chains corresponding to the phagemid library of recombinant peripheral blood lymphocyte immunoglobulin light chains of patients with systemic lupus erythematosus specifically hydrolyze only myelin basic protein (MBP). Canonical enzymes usually have only one active site catalyzing some kind of chemical reaction. It was shown previously that in contrast to classical enzymes, preparations of one of the light chains (NGTA2-Me-pro-Tr) showed two optimal pH values, two optimal concentrations of metal ions, and two Km values for MBP. One protease active site of NGTA2-Me-pro-Tr was trypsin like, whereas second one was metal dependent. In this article, a search for protein sequences of NGTA2-Me-pro-Tr responsible for catalytic functions was carried out. We performed, for the first time, analysis of the homology of the protein sequence of NGTA2-Me-pro-Tr with those of several classical Zn2+ - and Ca2+ -dependent, as well as human serine, proteases. The analysis allowed us to identify the protein sequences of NGTA2-Me-pro-Tr responsible for serine-like activity, the binding of MBP, and chelation of metal ions and catalysis directly. The data obtained are summarized using hypothetical models of the structure of the two active centers of a very unusual light chain of antibodies (Abs). The findings obtained may be very important for understanding possible structure of active centers of very unusual light chain of Abs possessing several enzymatic activities.

Selection and characterization of scFv antibody against nucleocapsid protein of Porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) is a widespread infectious agent in pigs. Nucleocapsid (N) protein of PRRSV has been identified as the most immunodominant viral protein. The main goal of the work was the selection and characterization of a single-chain antibody fragments (scFv) antibody specific to the N protein. Specific scFv antibody clone D5 was selected from the Tomlinson phagemid library and purified by immobilized metal affinity chromatography from the periplasmatic space of E. coli cells. The antibody was then characterized by sequencing and the ability to recognize the native virus N protein by Western blot and competitive ELISA. Pepscan analysis identified the position of the binding epitope between amino acids 62–84 of the N protein. Our study could help to improve the diagnostics and prevention of PRRSV in Central Europe.

Screening and purification of nanobodies from E. coli culture supernatants using the hemolysin secretion system

BackgroundThe hemolysin (Hly) secretion system of E. coli allows the one-step translocation of hemolysin A (HlyA) from the bacterial cytoplasm to the extracellular medium, without a periplasmic intermediate. In this work, we investigate whether the Hly secretion system of E. coli is competent to secrete a repertoire of functional single-domain VHH antibodies (nanobodies, Nbs), facilitating direct screening of VHH libraries and the purification of selected Nb from the extracellular medium.ResultsWe employed a phagemid library of VHHs obtained by immunization of a dromedary with three protein antigens from enterohemorrhagic E. coli (EHEC), namely, the extracellular secreted protein A (EspA), the extracellular C-terminal region of Intimin (Int280), and the translocated intimin receptor middle domain (TirM). VHH clones binding each antigen were enriched and amplified by biopanning, and subsequently fused to the C-terminal secretion signal of HlyA to be expressed and secreted in a E. coli strain carrying the Hly export machinery (HlyB, HlyD and TolC). Individual E. coli clones were grown and induced in 96-well microtiter plates, and the supernatants of the producing cultures directly used in ELISA for detection of Nbs binding EspA, Int280 and TirM. A set of Nb sequences specifically binding each of these antigens were identified, indicating that the Hly system is able to secrete a diversity of functional Nbs. We performed thiol alkylation assays demonstrating that Nbs are correctly oxidized upon secretion, forming disulphide bonds between cysteine pairs despite the absence of a periplasmic intermediate. In addition, we show that the secreted Nb-HlyA fusions can be directly purified from the supernatant of E. coli cultures, avoiding cell lysis and in a single affinity chromatography step.ConclusionsOur data demonstrate the Hly secretion system of E. coli can be used as an expression platform for screening and purification of Nb binders from VHH repertories.

Using Phage Display to Create Recombinant Antibodies.

A variety of phage display technologies have been developed since the approach was first described for antibodies. The most widely used approaches incorporate antibody sequences into the minor coat protein pIII of the nonlytic filamentous phage fd or M13. Libraries of variable gene sequences, encoding either scFv or Fab fragments, are made by incorporating sequences into phagemid vectors. The phagemid is packaged into phage particles with the assistance of a helper phage to produce the antibody display phage. This protocol describes a method for creating a phagemid library. The multiple cloning site (MCS) of the pBluescript KS(-) phagemid vector is replaced by digestion with the restriction enzyme BssHII, followed by the insertion of four overlapping oligonucleotides to create a new MCS within the vector. Next, the 3' portion of gene III (from M13mp18) is amplified and combined with an antibody sequence using overlap extension PCR. This product is inserted into the phagemid vector to create pPDS. Two helper plasmids are also created from the modified pBluescript vector: pLINK provides the linker between the heavy and light chains, and pFABC provides the CH1 domain of the heavy chain. An antibody cDNA library is constructed from the RNA of interest and ligated into pPDS. The phagemid library is electroporated into Escherichia coli cells along with the VCS-M13 helper phage.

Intracellular directed evolution of proteins from combinatorial libraries based on conditional phage replication

Directed evolution is a powerful tool to improve the characteristics of biomolecules. Here we present a protocol for the intracellular evolution of proteins with distinct differences and advantages in comparison with established techniques. These include the ability to select for a particular function from a library of protein variants inside cells, minimizing undesired coevolution and propagation of nonfunctional library members, as well as allowing positive and negative selection logics using basally active promoters. A typical evolution experiment comprises the following stages: (i) preparation of a combinatorial M13 phagemid (PM) library expressing variants of the gene of interest (GOI) and preparation of the Escherichia coli host cells; (ii) multiple rounds of an intracellular selection process toward a desired activity; and (iii) the characterization of the evolved target proteins. The system has been developed for the selection of new orthogonal transcription factors (TFs) but is capable of evolving any gene-or gene circuit function-that can be linked to conditional M13 phage replication. Here we demonstrate our approach using as an example the directed evolution of the bacteriophage λ cI TF against two synthetic bidirectional promoters. The evolved TF variants enable simultaneous activation and repression against their engineered promoters and do not cross-react with the wild-type promoter, thus ensuring orthogonality. This protocol requires no special equipment, allowing synthetic biologists and general users to evolve improved biomolecules within ∼7 weeks.

Production of Recombinant Human scFv Against Tetanus Toxin Heavy Chain by Phage Display Technology.

Tetanus, as a major cause of death in developing countries, is caused by tetanus neurotoxin. Recombinant antibodies against tetanus neurotoxin can be useful in tetanus management. Phage display of antibody fragments from immune human antibody libraries with single chain constructs combining the variable fragments (scFv) has been one of the most prominent technologies in antibody engineering. The aim of this study was the generation of a single chain fragment of variable region (scFv) library and selection of specific antibodies with high affinity against tetanus toxin. Immune human single chain fragment variable (HuscFv) antibody phagemid library was displayed on pIII of filamentous bacteriophage. Selection of scFv clones was performed against tetanus toxin antigens after three rounds of panning. The selected scFv clones were analyzed for inhibition of tetanus toxin binding to ganglioside GT1b. After the third round of panning, over 35 HuscFv phages specific for tetanus toxin were isolated from this library of which 15 clones were found to bind specifically to tetanus toxin. The selected HuscFv phages expressed as a soluble HuscFv peptide and some clones showed positive signals against tetanus toxin. We found that six HuscFv clones inhibit toxin binding to ganglioside GT1b. These selected antibodies can be used in the management of tetanus.

A new expression vector facilitating production and functional analysis of scFv antibody fragments selected from Tomlinson I + J phagemid libraries

Tomlinson I+J are synthetic phagemid human scFv libraries widely employed to obtain specific antibody fragments via a phage display method. The pIT2/HB2151 expression system proposed by the designers of the libraries has certain drawbacks which result in the lack of expression or low expression levels of numerous soluble scFvs. At the stage of scFv screening, this may lead to losing some excellent antibodies, which can be avoided but requires laborious and expensive work. Here we present a new, pET-30-based vector, which is compatible with Tomlinson libraries, retains all virtues of pIT2 used as a plasmid and eliminates all its flaws. We demonstrate that pET-scFv-T is frequently superior to pIT2 in terms of efficient scFv expression. Moreover, an amber suppressor bacterial strain, RosettaBlue(DE3)pLysS, transformed with the new vector, pET-scFv-T, coding for a number of scFvs, produces substantial amounts of functional, easy to purify recombinant antibody fragments, regardless of whether their coding sequences contain amber codons. Thus, pET-scFv-T/RosettaBlue(DE3)pLysS expression system seems to be a perfect tool for screening for the finest soluble scFvs selected from Tomlinson I+J, as well as from many other phagemid libraries.

Systemic lupus erythematosus: molecular cloning and analysis of recombinant monoclonal kappa light chain NGTA2-Me-pro-ChTr possessing two different activities-trypsin-like and metalloprotease.

Polyclonal antibodies hydrolyzing myelin basic protein (MBP) can play an important role in the pathogenesis of multiple sclerosis and systemic lupus erythematosus (SLE). An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of patients with SLE was used. The small pools of phage particles displaying light chains with different affinity for MBP were isolated by affinity chromatography on MBP-Sepharose. The fraction eluted with 0.5M NaCl was used for preparation of individual monoclonal light chains (MLChs, 26-27kDa). The clones were expressed in Escherichia coli in a soluble form; MLChs were purified by metal-chelating chromatography followed by gel filtration. In mammalians, there are serine proteases and metalloproteases. These and many other enzymes usually have only one active site and catalyze only one chemical reaction. In contrast to canonical proteases, one MLCh (NGTA2-Me-pro-ChTr) efficiently hydrolyzed MBP (but not other proteins) and four different oligopeptides corresponding to four immunodominant sequences containing cleavage sites of MBP. The proteolytic activity of MLCh was efficiently inhibited only by specific inhibitors of serine-like (phenylmethanesulfonylfluoride, PMSF) and metalloproteases (EDTA). It was shown that MLCh possess independent serine-like and metal-dependent activities. The principal existence of monoclonal antibodies with two different proteolytic activities is unexpected but very important for the further understanding of at present unknown biological functions of human antibodies.

Systemic lupus erythematosus: molecular cloning and analysis of 22 individual recombinant monoclonal kappa light chains specifically hydrolyzing human myelin basic protein.

Antibodies hydrolyzing myelin basic protein (MBP) can play an important role in the pathogenesis of multiple sclerosis (MS) and systemic lupus erythematosus (SLE). An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of patients with SLE was used. Small pools of phage particles displaying light chains with different affinities for MBP were isolated by affinity chromatography on MBP-Sepharose, and the fraction eluted with 0.5 M NaCl was used for preparation of individual monoclonal light chains (MLChs, 26-27 kDa). Seventy-two of 440 individual colonies were randomly chosen, expressed in Escherichia coli in a soluble form, and MLChs were purified by metal chelating chromatography. Twenty-two of 72 MLChs have high affinity and efficiently hydrolyze only MBP (not other control proteins) demonstrating various pH optima in a 5.7-9.0 range and different substrate specificity in the hydrolysis of four different MBP oligopeptides. Four MLChs demonstrated serine protease-like and three thiol protease-like activities, while 11 MLChs were metalloproteases. The activity of three MLChs was inhibited by both phenylmethylsulfonyl fluoride (PMSF) and Ethylenediaminetetraacetic acid (EDTA), two other by EDTA and iodoacetamide, and one by PMSF, EDTA, and iodoacetamide. The ratio of relative activity in the presence of Ca(2+), Mg(2+), Mn(2+), Ni(2+), Zn(2+), Cu(2+), and Co(2+) was individual for each of 22 MLCh preparations. It is the first examples of human MLChs, which probably can possess two or even three different proteolytic activities. These observations suggest an extreme diversity of anti-MBP abzymes in SLE patients. The immune systems of individual SLE patients can generate a variety of anti-MBP abzymes, which can attack MBP of myelin-proteolipid sheath of axons and play an important role in MS and SLE pathogenesis.

Novel Ubiquitin-derived High Affinity Binding Proteins with Tumor Targeting Properties

Targeting effector molecules to tumor cells is a promising mode of action for cancer therapy and diagnostics. Binding proteins with high affinity and specificity for a tumor target that carry effector molecules such as toxins, cytokines, or radiolabels to their intended site of action are required for these applications. In order to yield high tumor accumulation while maintaining low levels in healthy tissues and blood, the half-life of such conjugates needs to be in an optimal range. Scaffold-based binding molecules are small proteins with high affinity and short systemic circulation. Due to their low molecular complexity, they are well suited for combination with effector molecules as well as half-life extension technologies yielding therapeutics with half-lives adapted to the specific therapy. We have identified ubiquitin as an ideal scaffold protein due to its outstanding biophysical and biochemical properties. Based on a dimeric ubiquitin library, high affinity and specific binding molecules, so-called Affilin® molecules, have been selected against the extradomain B of fibronectin, a target almost exclusively expressed in tumor tissues. Extradomain B-binding molecules feature high thermal and serum stability as well as strong in vitro target binding and in vivo tumor accumulation. Application of several half-life extension technologies results in molecules of largely unaffected affinity but significantly prolonged in vivo half-life and tumor retention. Our results demonstrate the utility of ubiquitin as a scaffold for the generation of high affinity binders in a modular fashion, which can be combined with effector molecules and half-life extension technologies.