Improvement of an antibody neutralizing the anthrax toxin by simultaneous mutagenesis of its six hypervariable loops

The enhancement of antibody affinity by mutagenesis targeting only complementarity determining regions has the advantage of respecting the framework regions, which are important for tolerance if clinical use is envisaged. Here, starting from a Fab (antigen-binding fragment; 35PA₈₃) capable of neutralizing the lethal toxin of anthrax and having an affinity of 3.4 nM for its antigen, a phage-displayed library of variants where all six complementarity determining regions (73 positions) were targeted for mutagenesis was built. This library contained 5 × 10⁸ variants, and each variant carried four mutations on average. The library was first panned with adsorbed antigen and washes of increasing stringency. It was then screened in parallel with either small concentrations of soluble biotinylated antigen or adsorbed antigen and long elution times in the presence of soluble antigen. The stringencies of both selections were pushed as far as possible. Compared with 35PA₈₃, the best selected clone had an affinity enhanced 19-fold, to 180 pM, and its 50% inhibitory concentration was decreased by 40%. The results of the two selection methods were compared, and the generality of these methods was considered.     

De Novo Selection of High-Affinity Antibodies from Synthetic Fab Libraries Displayed on Phage as pIX Fusion Proteins

Filamentous phage was the first display platform employed to isolate antibodies in vitro and is still the most broadly used. The success of phage display is due to its robustness, ease of use, and comprehensive technology development, as well as a broad range of selection methods developed during the last two decades. We report here the first combinatorial synthetic Fab libraries displayed on pIX, a fusion partner different from the widely used pIII. The libraries were constructed on four V_L and three V_H domains encoded by IGV and IGJ germ-line genes frequently used in human antibodies, which were diversified to mirror the variability observed in the germ-line genes and antibodies isolated from natural sources. Two sets of libraries were built, one with diversity focused on V_H by keeping V_L in the germ-line gene configuration and the other with diversity in both V domains. After selection on a diverse panel of proteins, numerous specific Fabs with affinities ranging from 0.2 nM to 20 nM were isolated. V_H diversity was sufficient for isolating Fabs to most antigens, whereas variability in V_L was required for isolation of antibodies to some targets. After the application of an integrated maturation process consisting of reshuffling V_L diversity, the affinity of selected antibodies was improved up to 100-fold to the low picomolar range, suitable for in vivo studies. The results demonstrate the feasibility of displaying complex Fab libraries as pIX fusion proteins for antibody discovery and optimization and lay the foundation for studies on the structure-function relationships of antibodies.     

Human Framework Adaptation of a Mouse Anti-Human IL-13 Antibody

Humanization of a potent neutralizing mouse anti-human IL-13 antibody (m836) using a method called human framework adaptation (HFA) is reported. HFA consists of two steps: human framework selection (HFS) and specificity-determining residue optimization (SDRO). The HFS step involved generation of a library of m836 antigen binding sites combined with diverse human germline framework regions (FRs), which were selected based on structural and sequence similarities between mouse variable domains and a repertoire of human antibody germline genes. SDRO consisted of diversifying specificity-determining residues and selecting variants with improved affinity using phage display. HFS of m836 resulted in a 5-fold loss of affinity, whereas SDRO increased the affinity up to 100-fold compared to the HFS antibody. Crystal structures of Fabs in complex with IL-13 were obtained for m836, the HFS variant chosen for SDRO, and one of the highest-affinity SDRO variants. Analysis of the structures revealed that major conformational changes in FR-H1 and FR-H3 occurred after FR replacement, but none of them had an evident direct impact on residues in contact with IL-13. Instead, subtle changes affected the V_L/V_H (variable-light domain/variable-heavy domain) interface and were likely responsible for the 5-fold decreased affinity. After SDRO, increased affinity resulted mainly from rearrangements in hydrogen-bonding pattern at the antibody/antigen interface. Comparison with m836 putative germline genes suggested interesting analogies between natural affinity maturation and the engineering process that led to the potent HFA anti-human IL-13 antibody.     

Binding of N-terminal fragments of anthrax edema factor (EFN) and lethal factor (LFN) to the protective antigen pore

Anthrax toxin consists of three different molecules: the binding component protective antigen (PA, 83 kDa), and the enzymatic components lethal factor (LF, 90 kDa) and edema factor (EF, 89 kDa). The 63 kDa C-terminal part of PA, PA₆₃, forms heptameric channels that insert in endosomal membranes at low pH, necessary to translocate EF and LF into the cytosol of target cells. In many studies, about 30 kDa N-terminal fragments of the enzymatic components EF (254 amino acids) and LF (268 amino acids) were used to study their interaction with PA₆₃-channels. Here, in experiments with artificial lipid bilayer membranes, EF_N and LF_N show block of PA₆₃-channels in a dose, voltage and ionic strength dependent way with high affinity. However, when compared to their full-length counterparts EF and LF, they exhibit considerably lower binding affinity. Decreasing ionic strength and, in the case of EF_N, increasing transmembrane voltage at the cis side of the membranes, resulted in a strong decrease of half saturation constants. Our results demonstrate similarities but also remarkable differences between the binding kinetics of both truncated and full-length effectors to the PA₆₃-channel.     

Influenza PR8 HA-specific Fab fragments produced by phage display methods

Anti-influenza hemagglutinin (HA) Fabs were isolated from a phage display library using purified HA of influenza virus A/Puerto Rico/8/34 (PR8; H1N1) as an antigen. Four Fab clones displaying a 25-50-fold higher binding signal to PR8 HA than the control were selected for further analysis and comparison with anti-PR8 monoclonal antibody (mAb). All four Fabs and mAb recognized the PR8 HA under non-reducing conditions but rarely bound to reduced PR8 HA. Inhibition of influenza virus infection on MDCK cells was observed with Fab1 and mAb in a dose-dependent manner while Fab3 and 4 exhibited only a partial inhibitory effect. Moreover, Fab1 clone and mAb exhibited cross-reactivity with the A/Peking/262/95 (A/Peking; H1N1) strain. The inhibitory effects of mAb on both influenza strains were more potent than Fab1, which is likely attributed to its higher affinity for the antigen. SPR analyses, in fact, revealed that Fab1 and mAb have K_D of 1.5 × 10⁻⁸ and 3.2 × 10⁻⁹ M, respectively. These results strongly suggest that phage library-derived Fabs can be readily prepared and that such HA-specific Fabs with inhibitory action on influenza infection may be used to treat influenza patients.     

Effective delivery of antisense peptide nucleic acid oligomers into cells by anthrax protective antigen

Peptide nucleic acid (PNA) is highly stable and binds to complementary RNA and DNA with high affinity, but it resists cellular uptake, thereby limiting its bioavailability. We investigated whether protectiveantigen (PA, a non-toxic component of anthrax toxin) could transport antisense PNA oligomers into reporter cells that contain luciferase transgenes with mutant β-globin IVS2 intronic inserts, which permit aberrant pre-mRNA splicing and impair luciferase expression. PNA oligomers antisense to mutant splice sites in these IVS2 inserts induced luciferase expression when effectively delivered into the cells. PNA 18-mers with C-terminal poly-lysine tails [PNA(Lys)₈] demonstrated modest sequence-specific antisense activity by themselves at micromolar concentrations in luc-IVS2 reporter cell cultures. However, this activity was greatly amplified by PA. Antisense PNA(Lys)₈ with but not without PA also corrected the IVS2-654 β-globin splice defect in cultured erythroid precursor cells from a patient with β-thalassemia [genotype, IVS2-654(β⁰/β^E)], providing further evidence that anthrax PA can effectively transport antisense PNA oligomers into cells.     

Site-specific coupling and sterically controlled formation of multimeric antibody fab fragments with unnatural amino acids

Immunoconjugates and multispecific antibodies are rapidly emerging as highly potent experimental therapeutics against cancer. We have developed a method to incorporate an unnatural amino acid, p-acetylphenylalanine (pAcPhe) into an antibody antigen binding fragment (Fab) targeting HER2 (human epidermal growth factor receptor 2), allowing site-specific labeling without disrupting antigen binding. Expression levels of the pAcPhe-containing proteins were comparable to that of wild-type protein in shake-flask and fermentation preparations. The pAcPhe-Fabs were labeled by reaction with hydroxylamine dye and biotin species to produce well-defined, singly conjugated Fabs. We then coupled a hydroxylamine biotin to the pAcPhe-Fab and demonstrated controlled assembly of Fabs in the presence of the tetrameric biotin-binding protein, NeutrAvidin. The position of Fab biotinylation dictates the geometry of multimer assembly, producing unique multimeric Fab structures. These assembled Fab multimers differentially attenuate Her2 phosphorylation in breast cancer cells that overexpress the Her2 receptor. Thus, an encoded unnatural amino acid produces a chemical “handle” by which immunoconjugates and multimers can be engineered.     

The influence of antibody fragment format on phage display based affinity maturation of IgG

Today, most approved therapeutic antibodies are provided as immunoglobulin G (IgG), whereas small recombinant antibody formats are required for in vitro antibody generation and engineering during drug development. Particularly, single chain (sc) antibody fragments like scFv or scFab are well suited for phage display and bacterial expression, but some have been found to lose affinity during conversion into IgG.

In this study, we compared the influence of the antibody format on affinity maturation of the CD30-specific scFv antibody fragment SH313-F9, with the overall objective being improvement of the IgG. The variable genes of SH313-F9 were randomly mutated and then cloned into libraries encoding different recombinant antibody formats, including scFv, Fab, scFabΔC, and FabΔC. All tested antibody formats except Fab allowed functional phage display of the parental antibody SH313-F9, and the corresponding mutated antibody gene libraries allowed isolation of candidates with enhanced CD30 binding. Moreover, scFv and scFabΔC antibody variants retained improved antigen binding after subcloning into the single gene encoded IgG-like formats scFv-Fc or scIgG, but lost affinity after conversion into IgGs. Only affinity maturation using the Fab-like FabΔC format, which does not contain the carboxy terminal cysteines, allowed successful selection of molecules with improved binding that was retained after conversion to IgG. Thus, affinity maturation of IgGs is dependent on the antibody format employed for selection and screening. In this study, only FabΔC resulted in the efficient selection of IgG candidates with higher affinity by combination of Fab-like conformation and improved phage display compared with Fab.     

Microbioreactor Cultivations of Fab‐Producing Escherichia coli Reveal Genome‐Integrated Systems as Suitable for Prospective Studies on Direct Fab Expression Effects

Despite efforts to develop concepts for efficient antibody fragment (Fab) production in Escherichia coli (E. coli) and the high degree of similarity within this protein class, a generic platform technology is still not available. Indeed, feasible production of new Fab candidates remains challenging. In this study, a setup that enables direct characterization of host cell response to Fab expression by utilizing genome‐integrated (GI) systems is established. Among the multitude of factors that influence Fab expression, the variable domain, the translocation mechanism, the host strain, as well as the copy number of the gene of interest (GOI) are varied. The resulting 32 production clones are characterized in carbon‐limited microbioreactor cultivations with yields of 0–7.4 mg Fab per gram of cell dry mass. Antigen‐binding region variations have the greatest effect on Fab yield. In most cases, the E. coli HMS174(DE3) strain performs better than the BL21(DE3) strain. Translocation mechanism variations mainly influence leader peptide cleavage efficiency. Plasmid‐free systems, with a single copy of the GOI integrated into the chromosome, reach Fab yields in the range of 80–300% of plasmid‐based counterparts. Consequently, the GI Fab production clones could greatly facilitate direct analyses of systems response to different impact factors under varying production conditions.     

Human Germline Antibody Gene Segments Encode Polyspecific Antibodies

Structural flexibility in germline gene-encoded antibodies allows promiscuous binding to diverse antigens. The binding affinity and specificity for a particular epitope typically increase as antibody genes acquire somatic mutations in antigen-stimulated B cells. In this work, we investigated whether germline gene-encoded antibodies are optimal for polyspecificity by determining the basis for recognition of diverse antigens by antibodies encoded by three V_H gene segments. Panels of somatically mutated antibodies encoded by a common V_H gene, but each binding to a different antigen, were computationally redesigned to predict antibodies that could engage multiple antigens at once. The Rosetta multi-state design process predicted antibody sequences for the entire heavy chain variable region, including framework, CDR1, and CDR2 mutations. The predicted sequences matched the germline gene sequences to a remarkable degree, revealing by computational design the residues that are predicted to enable polyspecificity, i.e., binding of many unrelated antigens with a common sequence. The process thereby reverses antibody maturation in silico. In contrast, when designing antibodies to bind a single antigen, a sequence similar to that of the mature antibody sequence was returned, mimicking natural antibody maturation in silico. We demonstrated that the Rosetta computational design algorithm captures important aspects of antibody/antigen recognition. While the hypervariable region CDR3 often mediates much of the specificity of mature antibodies, we identified key positions in the V_H gene encoding CDR1, CDR2, and the immunoglobulin framework that are critical contributors for polyspecificity in germline antibodies. Computational design of antibodies capable of binding multiple antigens may allow the rational design of antibodies that retain polyspecificity for diverse epitope binding.     

Promoter engineering to optimize recombinant periplasmic Fab′ fragment production in Escherichia coli

Fab’ fragments have become an established class of biotherapeutic over the last two decades. Likewise, developments in synthetic biology are providing ever more powerful techniques for designing bacterial genes, gene networks and entire genomes that can be used to improve industrial performance of cells used for production of biotherapeutics. We have previously observed significant leakage of an exogenous therapeutic Fab’ fragment into the growth medium during high cell density cultivation of an Escherichia coli production strain. In this study we sought to apply a promoter engineering strategy to address the issue of Fab’ fragment leakage and its consequent bioprocess challenges. We used site directed mutagenesis to convert the P_tac promoter, present in the plasmid, pTTOD‐A33 Fab’, to a P_tic promoter which has been shown by others to direct expression at a 35% reduced rate compared to P_tac. We characterized the resultant production trains in which either P_tic or P_tac promoters direct Fab’ fragment expression. The P_tic promoter strain showed a 25?30% reduction in Fab’ expression relative to the original P_tac strain. Reduced Fab’ leakage and increased viability over the course of a fed‐batch fermentation were also observed for the P_tic promoter strain. We conclude that cell design steps such as the P_tac to P_tic promoter conversion reported here, can yield significant process benefit and understanding with respect to periplasmic Fab’ fragment production. It remains an open question as to whether the influence of transgene expression on periplasmic retention is mediated by global metabolic burden effects or periplasm overcapacity. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:840–847, 2016     

Structure of Fab fragment of malaria transmission blocking antibody 2A8 against P. vivax P25 protein

Understanding the structural basis of recognition between antigen and antibody requires the structural comparison of free and complexed components. Previously, we have reported the crystal structure of the complex between Fab fragment of murine monoclonal antibody 2A8 (Fab2A8) and Plasmodium vivax P25 protein (Pvs25) at 3.2 Å resolution. We report here the crystallization and X-ray structure of native Fab2A8 at 4.0 Å resolution. The 2A8 antibody generated against Pvs25 prevents the formation of P. vivax oocysts in the mosquito, when assayed in membrane feeding experiment.Comparison of native Fab2A8 structure with antigen bound Fab2A8 structure indicates the significant conformational changes in CDR-H1 and CDR-H3 regions of V_H domain and CDR-L3 region of V_L domain of Fab2A8. Upon complex formation, the relative orientation between V_L and V_H domains of Fab2A8 is conserved, while significant differences are observed in elbow angles of heavy and light chains. The combing site residues of complexed Fab2A8 exhibited the reduced temperature factor compared to native Fab2A8, suggesting a loss of conformational entropy upon antigen binding.     

Improved Protein-A separation of VH3 Fab from Fc after Papain Digestion of Antibodies

Antibody-binding fragments (Fab) are generated from whole antibodies by treatment with papain and can be separated from the Fc component using Protein-A affinity chromatography. Commercial kits are available, which facilitate the production and purification of Fab fragments; however, the manufacturer fails to report that this method is inefficient for antibodies with V_H3 domains as a result of the intrinsic variable region affinity for Protein-A. A commercially available, modified Protein-A resin (MabSelect SuRe) has been engineered for greater stability. Here, we report that an additional consequence of the modified resin is the ability to purify V_H3 family Fab fragments, which cannot be separated effectively from other components of the papain digest by traditional Protein-A resin. This improvement of a commonly used procedure is of significance, as increasingly, therapeutic antibodies are being derived from human origin, where V_H3 is the most abundantly used variable region family.     

Role of N-Terminal His6-Tags in Binding and Efficient Translocation of Polypeptides into Cells Using Anthrax Protective Antigen (PA)

It is of interest to define bacterial toxin biochemical properties to use them as molecular-syringe devices in order to deliver enzymatic activities into host cells. Binary toxins of the AB_7/8-type are among the most potent and specialized bacterial protein toxins. The B subunits oligomerize to form a pore that binds with high affinity host cell receptors and the enzymatic A subunit. This allows the endocytosis of the complex and subsequent injection of the A subunit into the cytosol of the host cells. Here we report that the addition of an N-terminal His₆-tag to different proteins increased their binding affinity to the protective antigen (PA) PA₆₃-channels, irrespective if they are related (C2I) or unrelated (gpJ, EDIN) to the AB_7/8-family of toxins. His₆-EDIN exhibited voltage-dependent increase of the stability constant for binding by a factor of about 25 when the trans-side corresponding to the cell interior was set to −70 mV. Surprisingly, the C. botulinum toxin C2II-channel did not share this feature of PA₆₃. Cell-based experiments demonstrated that addition of an N-terminal His₆-tag promoted also intoxication of endothelial cells by C2I or EDIN via PA₆₃. Our results revealed that addition of His₆-tags to several factors increase their binding properties to PA₆₃ and enhance the property to intoxicate cells.     

Generation and Characterization of High Affinity Humanized Fab Against Hepatitis B Surface Antigen

5S is a mouse monoclonal IgG1 that binds to the ‘a’ epitope of the Hepatitis B surface antigen (HBsAg) and tested positive in an in vitro test for virus neutralization. We have earlier reported the generation of humanized single chain variable fragment (scFv) from the same. In this article we report the generation of a recombinant Fab molecule by fusing humanized variable domains of 5S with the constant domains of human IgG1. The humanized Fab expressed in E. coli and subsequently purified, retained a high binding affinity (K_D = 3.63 nmol/L) to HBsAg and bound to the same epitope of HBsAg as the parent molecule. The humanized Fab also maintained antigen binding in the presence of various destabilizing agents like 3 M NaCl, 30% DMSO, 8 M urea, and extreme pH. This high affinity humanized Fab provides a basis for the development of therapeutic molecules that can be safely utilized for the prophylaxis and treatment for Hepatitis B infection.     

Use of high-capacity surface with oriented recombinant antibody fragments in a 5-min immunoassay for thyroid-stimulating hormone

High-capacity surfaces can enhance analyte-binding kinetics and be beneficial for rapid immunoassays. Site-specifically immobilized, oriented recombinant single-chain Fv (scFv) and Fab antibody fragments were compared with a conventional, nonoriented monoclonal antibody (Mab) to capture antigen from serum to solid surface in a one-step, two-site thyroid-stimulating hormone (TSH) immunoassay with a 5-min incubation time. The assay used a ready-to-use dry reagent-based concept and time-resolved fluorescent measurement. TSH binding capacities were 3.0-fold (Fab) and at least 4.1-fold (scFv) higher when recombinant antibodies were used instead of Mab. Recombinant antibody fragments also produced faster kinetics (5 vs. 45-min saturation level) than Mab: 21-25% (Mab) versus 72-83% (scFv and Fab). Analytical sensitivities of the 5-min assay were 0.09 mIU/L TSH (Fab), 0.16 mIU/L TSH (scFv), and 0.26 mIU/L TSH (Mab). Between-run variabilities were 4.2-7.9% (Fab), 4.6-17.7% (scFv), and 5.5-7.2% (Mab). The assays correlated well with the AutoDELFIA hTSH (human TSH) Ultra assay (r = 0.99, n = 109). Fab was good in all aspects of immunoassay—capacity, kinetics, sensitivity, and analytical performance. As a homogeneous, stable, and small-sized binding molecule with optimized surface-coating properties as well as reduced risk for interference by heterophilic antibodies, Fab fragment is a promising and realistic immunoreagent for the future.     

Generation, affinity maturation, and characterization of a human anti-human NKG2D monoclonal antibody with dual antagonistic and agonistic activity

In humans, NKG2D is an activating receptor on natural killer (NK) cells and a costimulatory receptor on certain T cells and plays a central role in mediating immune responses in autoimmune diseases, infectious diseases, and cancer. Monoclonal antibodies that antagonize or agonize immune responses mediated by human NKG2D are considered to be of broad and potent therapeutic utility. Nonetheless, monoclonal antibodies to NKG2D that are suitable for clinical investigations have not been published yet. Here, we describe the generation, affinity maturation, and characterization of a fully human monoclonal antibody to human NKG2D. Using phage display technology based on a newly generated naïve human Fab library in phage display vector pC3C followed by a tandem chain shuffling process designed for minimal deviation from natural human antibody sequences, we selected a human Fab, designated KYK-2.0, with high specificity and affinity to human NKG2D. KYK-2.0 Fab blocked the binding of the natural human NKG2D ligands MICA, MICB, and ULBP2 as potently as a commercially available mouse anti-human NKG2D monoclonal antibody in immunoglobulin G (IgG) format. Conversion of KYK-2.0 Fab to IgG1 resulted in subnanomolar avidity for human NKG2D. KYK-2.0 IgG1 was found to selectively recognize defined subpopulations of human lymphocytes known to express NKG2D, that is, the majority of human CD8+, CD16+, and CD56+ cells as well as a small fraction of human CD4+ cells. In solution, KYK-2.0 IgG1 interfered with the cytolytic activity of ex vivo expanded human NK cells. By contrast, immobilized KYK-2.0 IgG1 was found to strongly induce human NK cell activation. The dual antagonistic and agonistic activity promises a wide range of therapeutic applications for KYK-2.0 IgG1 and its derivatives.     

Loss of LIN-35, the Caenorhabditis elegans ortholog of the tumor suppressor p105Rb, results in enhanced RNA interference

Background

Genome-wide RNA interference (RNAi) screening is a very powerful tool for analyzing gene function in vivo in Caenorhabditis elegans. The effectiveness of RNAi varies from gene to gene, however, and neuronally expressed genes are largely refractive to RNAi in wild-type worms.

Results

We found that C. elegans strains carrying mutations in lin-35, the worm ortholog of the tumor suppressor gene p105Rb, or a subset of the genetically related synMuv B family of chromatin-modifying genes, show increased strength and penetrance for many germline, embryonic, and post-embryonic RNAi phenotypes, including neuronal RNAi phenotypes. Mutations in these same genes also enhance somatic transgene silencing via an RNAi-dependent mechanism. Two genes, mes-4 and zfp-1, are required both for the vulval lineage defects resulting from mutations in synMuv B genes and for RNAi, suggesting a common mechanism for the function of synMuv B genes in vulval development and in regulating RNAi. Enhanced RNAi in the germline of lin-35 worms suggests that misexpression of germline genes in somatic cells cannot alone account for the enhanced RNAi observed in this strain.

Conclusion

A worm strain with a null mutation in lin-35 is more sensitive to RNAi than any other previously described single mutant strain, and so will prove very useful for future genome-wide RNAi screens, particularly for identifying genes with neuronal functions. As lin-35 is the worm ortholog of the mammalian tumor suppressor gene p105Rb, misregulation of RNAi may be important during human oncogenesis.     

Dissecting the Alternatively Folded State of the Antibody Fab Fragment

Intact antibodies and antigen binding fragments (Fab) have been previously shown to form an alternatively folded state (AFS) at low pH. This state consists primarily of secondary structure interactions, with reduced tertiary structure content. The AFS can be distinguished from the molten globule state by the formation of nonnative structure and, in particular, its high stability. In this study, the isolated domains of the MAK33 (murine monoclonal antibody of the subtype κ/IgG1) Fab fragment were investigated under conditions that have been reported to induce the AFS. Surprising differences in the ability of individual domains to form the AFS were observed, despite the similarities in their native structures. All Fab domains were able to adopt the AFS, but only for V_H (variable domain of the heavy chain) could a significant amount of tertiary structure be detected and different conditions were needed to induce the AFS. V_H, the least stable of the domains under physiological conditions, was the most stable in the AFS, yet all domains showed significant stability against thermal and chemical unfolding in their AFS. Formation of the AFS was found to generally proceed via the unfolded state, with similar rates for most of the domains. Taken together, our data reveal striking differences in the biophysical properties of the AFS of individual antibody domains that reflect the variation possible for domains of highly homologous native structures. Furthermore, they allow individual domain contributions to be dissected from specific oligomer effects in the AFS of the antibody Fab fragment.