ATP hydrolysis provides functions that promote rejection of pairings between different copies of long repeated sequences.

During DNA recombination and repair, RecA family proteins must promote rapid joining of homologous DNA. Repeated sequences with >100 base pair lengths occupy more than 1% of bacterial genomes; however, commitment to strand exchange was believed to occur after testing ∼20-30 bp. If that were true, pairings between different copies of long repeated sequences would usually become irreversible. Our experiments reveal that in the presence of ATP hydrolysis even 75 bp sequence-matched strand exchange products remain quite reversible. Experiments also indicate that when ATP hydrolysis is present, flanking heterologous dsDNA regions increase the reversibility of sequence matched strand exchange products with lengths up to ∼75 bp. Results of molecular dynamics simulations provide insight into how ATP hydrolysis destabilizes strand exchange products. These results inspired a model that shows how pairings between long repeated sequences could be efficiently rejected even though most homologous pairings form irreversible products.

ssDNA Pairing Accuracy Increases When Abasic Sites Divide Nucleotides into Small Groups.

Accurate sequence dependent pairing of single-stranded DNA (ssDNA) molecules plays an important role in gene chips, DNA origami, and polymerase chain reactions. In many assays accurate pairing depends on mismatched sequences melting at lower temperatures than matched sequences; however, for sequences longer than ~10 nucleotides, single mismatches and correct matches have melting temperature differences of less than 3°C. We demonstrate that appropriately grouping of 35 bases in ssDNA using abasic sites increases the difference between the melting temperature of correct bases and the melting temperature of mismatched base pairings. Importantly, in the presence of appropriately spaced abasic sites mismatches near one end of a long dsDNA destabilize the annealing at the other end much more effectively than in systems without the abasic sites, suggesting that the dsDNA melts more uniformly in the presence of appropriately spaced abasic sites. In sum, the presence of appropriately spaced abasic sites allows temperature to more accurately discriminate correct base pairings from incorrect ones.

Tension on dsDNA bound to ssDNA-RecA filaments may play an important role in driving efficient and accurate homology recognition and strand exchange.

It is well known that during homology recognition and strand exchange the double stranded DNA (dsDNA) in DNA/RecA filaments is highly extended, but the functional role of the extension has been unclear. We present an analytical model that calculates the distribution of tension in the extended dsDNA during strand exchange. The model suggests that the binding of additional dsDNA base pairs to the DNA/RecA filament alters the tension in dsDNA that was already bound to the filament, resulting in a non-linear increase in the mechanical energy as a function of the number of bound base pairs. This collective mechanical response may promote homology stringency and underlie unexplained experimental results.

Back to the future: recombinant polyclonal antibody therapeutics.

Antibody therapeutics are one of the fastest growing classes of pharmaceuticals, with an annual US market over $20 billion, developed to treat a variety of diseases including cancer, auto-immune and infectious diseases. Most are currently administered as a single molecule to treat a single disease, however there is mounting evidence that cocktails of multiple antibodies, each with a unique binding specificity and protective mechanism, may improve clinical efficacy. Here, we review progress in the development of oligoclonal combinations of antibodies to treat disease, focusing on identification of synergistic antibodies. We then discuss the application of modern antibody engineering technologies to produce highly potent antibody preparations, including oligoclonal antibody cocktails and truly recombinant polyclonal antibodies. Specific examples illustrating the synergy conferred by multiple antibodies will be provided for diseases caused by botulinum toxin, cancer and immune thrombocytopenia. The bioprocessing and regulatory options for these preparations will be discussed.

Complementary strand relocation may play vital roles in RecA-based homology recognition.

RecA-family proteins mediate homologous recombination and recombinational DNA repair through homology search and strand exchange. Initially, the protein forms a filament with the incoming single-stranded DNA (ssDNA) bound in site I. The RecA-ssDNA filament then binds double-stranded DNA (dsDNA) in site II. Non-homologous dsDNA rapidly unbinds, whereas homologous dsDNA undergoes strand exchange yielding heteroduplex dsDNA in site I and the leftover outgoing strand in site II. We show that applying force to the ends of the complementary strand significantly retards strand exchange, whereas applying the same force to the outgoing strand does not. We also show that crystallographically determined binding site locations require an intermediate structure in addition to the initial and final structures. Furthermore, we demonstrate that the characteristic dsDNA extension rates due to strand exchange and free RecA binding are the same, suggesting that relocation of the complementary strand from its position in the intermediate structure to its position in the final structure limits both rates. Finally, we propose that homology recognition is governed by transitions to and from the intermediate structure, where the transitions depend on differential extension in the dsDNA. This differential extension drives strand exchange forward for homologs and increases the free energy penalty for strand exchange of non-homologs.

RecA homology search is promoted by mechanical stress along the scanned duplex DNA.

A RecA-single-stranded DNA (RecA-ssDNA) filament searches a genome for sequence homology by rapidly binding and unbinding double-stranded DNA (dsDNA) until homology is found. We demonstrate that pulling on the opposite termini (3' and 5') of one of the two DNA strands in a dsDNA molecule stabilizes the normally unstable binding of that dsDNA to non-homologous RecA-ssDNA filaments, whereas pulling on the two 3', the two 5', or all four termini does not. We propose that the 'outgoing' strand in the dsDNA is extended by strong DNA-protein contacts, whereas the 'complementary' strand is extended by the tension on the base pairs that connect the 'complementary' strand to the 'outgoing' strand. The stress resulting from different levels of tension on its constitutive strands causes rapid dsDNA unbinding unless sufficient homology is present.

Changes in the tension in dsDNA alter the conformation of RecA bound to dsDNA-RecA filaments.

The RecA protein is an ATPase that mediates recombination via strand exchange. In strand exchange a single-stranded DNA (ssDNA) bound to RecA binding site I in a RecA/ssDNA filament pairs with one strand of a double-stranded DNA (dsDNA) and forms heteroduplex dsDNA in site I if homology is encountered. Long sequences are exchanged in a dynamic process in which initially unbound dsDNA binds to the leading end of a RecA/ssDNA filament, while heteroduplex dsDNA unbinds from the lagging end via ATP hydrolysis. ATP hydrolysis is required to convert the active RecA conformation, which cannot unbind, to the inactive conformation, which can unbind. If dsDNA extension due to RecA binding increases the dsDNA tension, then RecA unbinding must decrease tension. We show that in the presence of ATP hydrolysis decreases in tension induce decreases in length whereas in the absence of hydrolysis, changes in tension have no systematic effect. These results suggest that decreases in force enhance dissociation by promoting transitions from the active to the inactive RecA conformation. In contrast, increases in tension reduce dissociation. Thus, the changes in tension inherent to strand exchange may couple with ATP hydrolysis to increase the directionality and stringency of strand exchange.

Study of force induced melting of dsDNA as a function of length and conformation.

We measure the constant force required to melt double-stranded (ds) DNA as a function of length for lengths from 12 to 100,000 base pairs, where the force is applied to the 3'3' or 5'5' ends of the dsDNA. Molecules with 32 base pairs or fewer melt before overstretching. For these short molecules, the melting force is independent of the ends to which the force is applied and the shear force as a function of length is well described by de Gennes theory with a de Gennes length of less than 10 bp. Molecules with lengths of 500 base pairs or more overstretch before melting. For these long molecules, the melting force depends on the ends to which the force is applied. The melting force as a function of length increases even when the length exceeds 1000 bp, where the length dependence is inconsistent with de Gennes theory. Finally, we expand de Gennes melting theory to 3'5' pulling and compare the predictions with experimental results.

The structure of DNA overstretched from the 5'5' ends differs from the structure of DNA overstretched from the 3'3' ends.

It has been suggested that the structure that results when double-stranded DNA (dsDNA) is pulled from the 3'3' ends differs from that which results when it is pulled from the 5'5' ends. In this work, we demonstrate, using lambda phage dsDNA, that the overstretched states do indeed show different properties, suggesting that they correspond to different structures. For 3'3' pulling versus 5'5' pulling, the following differences are observed: (i) the forces at which half of the molecules in the ensemble have made a complete force-induced transition to single stranded DNA are 141 +/- 3 pN and 122 +/- 4 pN, respectively; (ii) the extension vs. force curve for overstretched DNA has a marked change in slope at 127 +/- 3 pN for 3'3' and 110 +/- 3 pN for 5'5'; (iii) the hysteresis (H) in the extension vs. force curves at 150 mM NaCl is 0.3 +/- 0.8 pN microm for 3'3' versus 13 +/- 8 pN for 5'5'; and (iv) 3'3' and 5'5' molecules show different changes in hysteresis due to interactions with beta-cyclodextrin, a molecule that is known to form stable host-guest complexes with rotated base pairs, and glyoxal that is known to bind stably to unpaired bases. These differences and additional findings are well-accommodated by the corresponding structures predicted on theoretical grounds.

Probing the mechanical stability of DNA in the presence of monovalent cations.

We examine the interaction between monovalent cations and DNA using several different assays that measure the stability of double-stranded DNA (dsDNA). The thermal melting of dsDNA and the mechanical separation of dsDNA into two single strands both depend on the stability of dsDNA with respect to ssDNA and are sensitive to the interstrand phosphate repulsion. We find that the experimentally measured melting temperatures and unzipping forces are approximately the same for all of the ions considered in this study. Likewise, the force required to transform B-DNA into the overstretched form is also similar for all of the ions. In contrast, for a given ion concentration, the force at which the overstretched state fully relaxes back to the canonical B-DNA form depends on the cation; however, for all cations, the overstretching force decreases with decreasing ion concentration, suggesting that this force is sensitive to screening. We observe a general trend for smaller ions to produce more efficient relaxation. Finally, for a given cation, the relaxation can also depend on the anion.

Extensive restrictions in the VH sequence usage of the human antibody response against the Rhesus D antigen.

Anti-Rhesus D immunoglobulin purified from human sera is used as a prophylactic reagent in Rhesus D negative women at risk of alloimmunization during pregnancy. We are currently developing a Rhesus D antigen-specific recombinant polyclonal antibody drug lead for replacing the existing blood derived-products. By analyzing the RhD-specific antibody VH repertoires from eight alloimmunized women we found, in agreement with previous studies, a strong preference for the VH 3-33 "superspecies" gene segments which encompasses the IGHV3-30-3*01, IGHV3-30*18, and IGHV3-33*01 VH alleles. Even more extensive genetic restriction was observed among five donors, which produced antibodies of identical V-D-J usage and CDR3 loop length and joining regions of similar amino acid composition. In addition, we find a high degree of sequence relatedness to previously isolated anti-Rhesus D antibodies. Such close homology in VH domains indicates that significant structural restrictions are operating in the selection of antibodies recognizing RhD as seen for T cell receptors. Moreover, some VH domains were isolated in their germline configuration indicating that anti-RhD antibodies of relatively high affinity are present in the naïve antibody repertoire of Rhesus negative individuals which offers an explanation for the strong and clinically significant immunogenicity of the Rhesus D.

Reduced susceptibility of recombinant polyclonal antibodies to inhibitory anti-variable domain antibody responses.

The immunogenicity of therapeutic Abs is a concern as anti-drug Abs may impact negatively on the pharmacodynamics and safety profile of Ab drugs. The factors governing induction of anti-drug Abs are not fully understood. In this study, we describe a model based on mouse-human chimeric Abs for the study of Ab immunogenicity in vivo. Six chimeric Abs containing human V regions and mouse C regions were generated from six human anti-Rhesus D Abs and the Ag-binding characteristics of the parental human Abs were retained. Analysis of the immune response toward the individual chimeric Abs revealed the induction of anti-variable domain Abs including anti-idiotypic Abs against some of these, thereby demonstrating the applicability of the model for studying anti-drug Ab responses in vivo. Immunization of BALB/c, C57, and outbred NMRI mice with a polyclonal composition consisting of all six chimeric Abs demonstrated that the immunogenicity of the individual Abs was haplotype dependent. Chimeric Abs, which were nonimmunogenic when administered individually, did not become immunogenic as part of the polyclonal composition, implying the absence of epitope spreading. Ex vivo Ab-binding studies established a clear correlation between the level of immunogenicity of the Abs comprised in the composition and the impact on the pharmacology of the Abs. These analyses demonstrate that under these conditions this polyclonal Ab composition was generally less susceptible to blocking Abs than the respective mAbs.

Production of target-specific recombinant human polyclonal antibodies in mammalian cells.

We describe the expression and consistent production of a first target-specific recombinant human polyclonal antibody. An anti-Rhesus D recombinant polyclonal antibody, Sym001, comprised of 25 unique human IgG1 antibodies, was produced by the novel Sympress expression technology. This strategy is based on site-specific integration of antibody genes in CHO cells, using the FRT/Flp-In recombinase system. This allows integration of the expression construct at the same genomic site in the host cells, thereby reducing genomic position effects. Different bioreactor batches of Sym001 displayed highly consistent manufacturing yield, antibody composition, binding potency, and functional activity. The results demonstrate that diverse recombinant human polyclonal antibody compositions can be reproducibly generated under conditions directly applicable to industrial manufacturing settings and present a first recombinant polyclonal antibody which could be used for treatment of hemolytic disease of the newborn and/or idiopathic thrombocytopenic purpura.

Isolation of human antibody repertoires with preservation of the natural heavy and light chain pairing.

The humoral immune system in higher vertebrates is unique in its ability to generate highly diverse antibody responses against most pathogens as well as against certain malignancies. Several technologies have been developed to exploit this vast source of potentially therapeutic antibodies, including hybridoma technology, phage display and yeast display. Here, we present a novel, high-throughput technology (the Symplex Technology) for rapid direct cloning and identification of human antigen-specific high-affinity antibodies from single antibody-producing cells of immune individuals. The utility of the technology was demonstrated by isolation of diverse sets of unique high-affinity antibodies against tetanus toxoid and influenza virus from immunized volunteers. Hence, the Symplex Technology is a new method for the rapid isolation of high-affinity antibodies directly from humans.

DNA unzipped under a constant force exhibits multiple metastable intermediates.

Single molecule studies, at constant force, of the separation of double-stranded DNA into two separated single strands may provide information relevant to the dynamics of DNA replication. At constant applied force, theory predicts that the unzipped length as a function of time is characterized by jumps during which the strands separate rapidly, followed by long pauses where the number of separated base pairs remains constant. Here, we report previously uncharacterized observations of this striking behavior carried out on a number of identical single molecules simultaneously. When several single lambda phage molecules are subject to the same applied force, the pause positions are reproducible in each. This reproducibility shows that the positions and durations of the pauses in unzipping provide a sequence-dependent molecular fingerprint. For small forces, the DNA remains in a partially unzipped state for at least several hours. For larger forces, the separation is still characterized by jumps and pauses, but the double-stranded DNA will completely unzip in less than 30 min.