Antigen retrieval in cells and tissues: enhancement with sodium dodecyl sulfate

Immunocytochemistry provides important information on the localization of antigens in cells and tissues. However, the procedures used to prepare cells and tissues for immunocytochemical labeling may have deleterious effects on the results achieved. That is, the antigen of interest may be difficult or impossible to detect following labeling. These sorts of observations have led to the concept of antigen masking in which the antigen (or specific epitope) is hidden from antibodies specific for that antigen (or epitope). Various procedures to circumvent this problem have been developed. These different procedures generally fit under the term "antigen retrieval" (or epitope retrieval). The practice of antigen retrieval is widely employed with paraffin-embedded material. Antigen retrieval is less often applied to cells and tissues that are not embedded in paraffin. However, in the latter preparations there are situations in which the observed immunolabeling achieved falls short of expectations. This poor level of immunolabeling may, in some situations, be improved upon with antigen retrieval procedures. In this review, we describe experimental situations in which immunolabeling fell short of expectations. We also describe a procedure that has been useful in enhancing immunolabeling efficiency in these cases. The major feature of this procedure is the incorporation of a permeabilization/denaturation step using sodium dodecyl sulfate. This postfixation and prelabeling step dramatically improves immunolabeling for a number of antigens in both cells and cryosections of tissue.     

Phage display screening of TIGIT-specific antibody for antitumor immunotherapy

ABSTRACT

The fully synthetic humanized phage antibody library has the advantages including the minimized immunogenicity, which frequently happened in hybridoma cell-based antibody production. In this paper, using the constructed diverse complementarity determining region gene library and the germline gene as the backbone, we constructed eight single-chain antibody libraries and a combinatorial antibody library with a big capacity of 1.41 × 10¹⁰. M13EEA helper phage that was engineered from M13KO7 was applied to prepare phage antibody library. The eukaryotic expression of T-cell immune receptor with Ig and ITIM domain (TIGIT) antigen was used as a target antigen for screening. The screening of antigen-specific single-chain Fc-fused protein was performed through evaluation of binding affinity based on ELISA analysis. The IgG antibody was prepared with the screened single-chain protein. Finally, the CB3 antibody was screened out which exhibits the highest binding affinity with TIGIT with the K_d value of 8.155 × 10^?10 M.     

Characterizing and Diminishing Autofluorescence in Formalin-fixed Paraffin-embedded Human Respiratory Tissue

Tissue autofluorescence frequently hampers visualization of immunofluorescent markers in formalin-fixed paraffin-embedded respiratory tissues. We assessed nine treatments reported to have efficacy in reducing autofluorescence in other tissue types. The three most efficacious were Eriochrome black T, Sudan black B and sodium borohydride, as measured using white light laser confocal Λ² (multi-lambda) analysis. We also assessed the impact of steam antigen retrieval and serum application on human tracheal tissue autofluorescence. Functionally fitting this Λ² data to 2-dimensional Gaussian surfaces revealed that steam antigen retrieval and serum application contribute minimally to autofluorescence and that the three treatments are disparately efficacious. Together, these studies provide a set of guidelines for diminishing autofluorescence in formalin-fixed paraffin-embedded human respiratory tissue. Additionally, these characterization techniques are transferable to similar questions in other tissue types, as demonstrated on frozen human liver tissue and paraffin-embedded mouse lung tissue fixed in different fixatives.     

A multispecific monoclonal antibody G2 recognizes at least three completely different epitope sequences with high affinity

A monoclonal antibody (mAb) G2 possesses an unusual characteristic of reacting with at least three proteins (ATP6V1C1, SEPT3, and C6H10orf76) other than its original antigen, chicken prion protein (ChPrP). The epitopes on ChPrP and ATP6V1C1 have been identified previously. In this study, we identified the epitope in the third protein, SEPT3. Interestingly, there was no amino acid sequence similarity among the epitopes on the three proteins. These epitopes had high binding affinities to G2 (K _D = ～10^?7 M for monovalent binding and K _D = ～10^?9 M for divalent binding), as determined using a SPR biosensor. This is the first report on a three‐in‐one mAb recognizing completely different epitope sequences with high affinity. Additionally, competitive ELISA indicated that the binding sites on G2, specific for the three different epitopes, overlapped, suggesting that the antigen‐binding site may be flexible in the free form and capable of adapting to at least three different conformations to enable interactions with three different antigens.     

OptMAVEn – A New Framework for the de novo Design of Antibody Variable Region Models Targeting Specific Antigen Epitopes

Antibody-based therapeutics provides novel and efficacious treatments for a number of diseases. Traditional experimental approaches for designing therapeutic antibodies rely on raising antibodies against a target antigen in an immunized animal or directed evolution of antibodies with low affinity for the desired antigen. However, these methods remain time consuming, cannot target a specific epitope and do not lead to broad design principles informing other studies. Computational design methods can overcome some of these limitations by using biophysics models to rationally select antibody parts that maximize affinity for a target antigen epitope. This has been addressed to some extend by OptCDR for the design of complementary determining regions. Here, we extend this earlier contribution by addressing the de novo design of a model of the entire antibody variable region against a given antigen epitope while safeguarding for immunogenicity (Optimal Method for Antibody Variable region Engineering, OptMAVEn). OptMAVEn simulates in silico the in vivo steps of antibody generation and evolution, and is capable of capturing the critical structural features responsible for affinity maturation of antibodies. In addition, a humanization procedure was developed and incorporated into OptMAVEn to minimize the potential immunogenicity of the designed antibody models. As case studies, OptMAVEn was applied to design models of neutralizing antibodies targeting influenza hemagglutinin and HIV gp120. For both HA and gp120, novel computational antibody models with numerous interactions with their target epitopes were generated. The observed rates of mutations and types of amino acid changes during in silico affinity maturation are consistent with what has been observed during in vivo affinity maturation. The results demonstrate that OptMAVEn can efficiently generate diverse computational antibody models with both optimized binding affinity to antigens and reduced immunogenicity.     

A Novel Antibody Humanization Method Based on Epitopes Scanning and Molecular Dynamics Simulation

1-17-2 is a rat anti-human DEC-205 monoclonal antibody that induces internalization and delivers antigen to dendritic cells (DCs). The potentially clinical application of this antibody is limited by its murine origin. Traditional humanization method such as complementarity determining regions (CDRs) graft often leads to a decreased or even lost affinity. Here we have developed a novel antibody humanization method based on computer modeling and bioinformatics analysis. First, we used homology modeling technology to build the precise model of Fab. A novel epitope scanning algorithm was designed to identify antigenic residues in the framework regions (FRs) that need to be mutated to human counterpart in the humanization process. Then virtual mutation and molecular dynamics (MD) simulation were used to assess the conformational impact imposed by all the mutations. By comparing the root-mean-square deviations (RMSDs) of CDRs, we found five key residues whose mutations would destroy the original conformation of CDRs. These residues need to be back-mutated to rescue the antibody binding affinity. Finally we constructed the antibodies in vitro and compared their binding affinity by flow cytometry and surface plasmon resonance (SPR) assay. The binding affinity of the refined humanized antibody was similar to that of the original rat antibody. Our results have established a novel method based on epitopes scanning and MD simulation for antibody humanization.     

Highly specific off-target binding identified and eliminated during the humanization of an antibody against FGF receptor 4

Off-target binding can significantly affect the pharmacokinetics (PK), tissue distribution, efficacy and toxicity of a therapeutic antibody. Herein we describe the development of a humanized anti-fibroblast growth factor receptor 4 (FGFR4) antibody as a potential therapeutic for hepatocellular carcinoma (HCC). A chimeric anti-FGFR4 monoclonal antibody (chLD1) was previously shown to block ligand binding and to inhibit FGFR4-mediated signaling as well as tumor growth in vivo. A humanized version of chLD1, hLD1.vB, had similar binding affinity and in vitro blocking activity, but it exhibited rapid clearance, poor target tissue biodistribution and limited efficacy when compared to chLD1 in a HUH7 human HCC xenograft mouse model. These problems were traced to instability of the molecule in rodent serum. Size exclusion high performance liquid chromatography, immunoprecipitation and mass spectral sequencing identified a specific interaction between hLD1.vB and mouse complement component 3 (C3). A PK study in C3 knock-out mice further confirmed this specific interaction. Subsequently, an affinity-matured variant derived from hLD1.vB (hLD1.v22), specifically selected for its lack of binding to mouse C3 was demonstrated to have a PK profile and in vivo efficacy similar to that of chLD1 in mice. Although reports of non-specific off-target binding have been observed for other antibodies, this represents the first report identifying a specific off-target interaction that affected disposition and biological activity. Screens developed to identify general non-specific interactions are likely to miss the rare and highly specific cross-reactivity identified in this study, thus highlighting the importance of animal models as a proxy for avoiding unexpected clinical outcomes.Key words: antibody humanization, non-specific binding, fibroblast growth factor receptor 4, affinity maturation, off-target binding, complement C3Antibodies are an attractive source of biotherapeutic agents due to their high affinity, exquisite target selectivity and extended half-life in vivo. Their development for therapeutic applications has been facilitated by hybridoma technology, antibody humanization and numerous in vitro antibody selection technologies that enable antibodies with desired biological properties to be engineered at will.Co-incident with this increase in the in vitro development of antibodies for therapeutic applications has been the recognition of how antibodies evolve in vivo. Several studies have pointed to the relationship between antibody affinity and antigen specificity.^1–6 The conformational flexibility of initial recombined antibodies is considered to be an important feature of the immune system''s ability to generate antibodies against a broad spectrum of antigens. During antibody maturation, this structural plasticity is thought to be restricted through somatic hypermutation in vivo (and perhaps affinity maturation in vitro) leading to a reduced entropy cost for specific antigen binding and a corresponding increase in antigen specificity. The increase in antigen specificity helps to eliminate undesired off-target antibody interactions, and serves as part of immune system checkpoints designed to prevent autoimmune disease.In contrast, antibodies that are generated in vitro lack any regulatory immune surveillance. For these, various screens utilizing protein chips and microarrays have been developed in order to evaluate or anticipate off-target interactions.^7–9 In one study comparing antibodies against TNFα, for example, multiple off-target interactions were found for adalimumab, an antibody derived in vitro from a cloned human antibody phage library.⁷ However, such screens are artificial and whether any observed non-specific off-target binding events result in adverse side effects in vivo or actually take place in vivo has yet to be demonstrated.Recently, affinity matured variants of palivizumab were found to have a less than anticipated increase in potency as a prophylactic treatment in a rat model of respiratory syncytial virus (RSV). The variants unexpectedly exhibited broad tissue binding that led to their rapid clearance and low target tissue bioavailability.¹⁰ Reversion of some of the amino acid changes incorporated during affinity maturation diminished much of the non-specific tissue binding and improved efficacy and pharmacokinetics. Direct interactions leading to the broad non-specific tissue binding were thus identified and removed, enabling the development of a more effective variant of palivizumab. This is the first report correlating broad tissue cross-reactivity in vitro with rapid clearance and poor tissue bioavailability.The likelihood of identifying an off-target binding event is a function of the size of the protein repertoire and the affinity that is considered relevant.¹¹ Given the huge complexity of an in vivo system and the typically high therapeutic dosing in a clinical setting, the odds of an off-target event that affects efficacy, clearance, tissue bioavailability or toxicity are greatly increased. Such antigen promiscuity in an antibody may arise from the recognition of structurally related epitopes (molecular mimicry), the utilization of overlapping or independent antibody paratopes or through conformational flexibility that enables the complementary-determining regions (CDRs) or side chains of an antibody to adapt to more than one antigen.^12,13In contrast to the broad non-specific off-target recognition and antibody polyspecificity described above, here we report an unexpected specific off-target binding event we identified for a humanized antibody directed toward fibroblast growth factor receptor (FGFR) 4. The FGFR signaling system plays critical roles in a variety of normal developmental and physiological processes, and aberrant signaling may lead to tumor development and progression.¹⁴ FGFR4 has been shown to play a modulatory role in the development and progression of hepatocellular carcinoma (HCC) in mice, and potentially in humans. A chimeric anti-FGFR4 monoclonal antibody (chLD1) was previously shown to block ligand binding, inhibit FGFR4-mediated signaling and inhibit HCC tumor growth in vivo.¹⁴ Following the humanization of chLD1, we observed an unexpected loss of efficacy in a mouse tumor model, although the humanized variant had identical affinity for FGFR4. This variant was found to cross-react with an abundant mouse serum protein. This specific off-target interaction interfered with FGFR4 binding, altered antibody clearance, impacted target tissue distribution, resulting in reduced therapeutic activity. Subsequently we eliminated this off-target binding through affinity maturation of the humanized antibody leading to the full restoration of the in vivo properties inherent in chLD1.This work illustrates some of the challenges that extend well beyond simple antibody-antigen binding and serves as a cautionary tale to therapeutic antibody development.     

Repetitive versus monomeric antigen presentation: direct visualization of antibody affinity and specificity

The concept of presenting antigens in a repetitive array to obtain high titers of specific antibodies is increasingly applied by using surface-engineered viruses or bacterial envelopes as novel vaccines. A case for this concept was made 25 years ago, when producing high-titer antisera against ordered arrays of gp23, the major capsid protein of bacteriophage T4 (Aebi et al., Proc. Natl. Acad. Sci. USA, 74 (1977) 5514-5518). In view of the current interest in this concept we thought it useful to employ this system to directly visualize the dependence of antibody affinity and specificity on antigen presentation. We compared antibodies raised against T4 polyheads, a tubular variant of the bacteriophage T4 capsid, which have gp23 hexamers arranged in a crystalline lattice (gp23(repetitive)), with those raised against the hexameric gp23 subunits (gp23(monomeric)). The labeling patterns of Fab-fragments prepared from these antibodies when bound to polyheads were determined by electron microscopy and image enhancement. Anti-gp23(repetitive) bound in a monospecific, stoichiometric fashion to the gp23 units constituting the polyhead surface. In contrast, anti-gp23(monomeric) decorated the polyhead surface randomly and with a 40-fold lower occupancy. These results concur with the difference in titers established by ELISA for the antisera against the repetitively displayed form of antigen (anti-gp23(repetitive)) and the randomly presented antigen (gp23(monomeric)), and they constitute a compelling visual documentation of the concept of repetitive antigen presentation to elicite a serotype-like immune response.     

Synthesis and preliminary in vitro evaluation of DOTA-Tenatumomab conjugates for theranostic applications in tenascin expressing tumors

Tenatumomab is an anti-tenascin murine monoclonal antibody previously used in clinical trials for delivering radionuclides to tumors by both pre-targeting (biotinylated Tenatumomab within PAGRIT) and direct ¹³¹Iodine labeling approaches. Here we present the synthesis and in vitro characterization of three Tenatumomab conjugates to bifunctional chelating agents (NHS-DOTA, NCS-DOTA and NCS-DTPA). Results indicate ST8198AA1 (Tenatumomab-DOTAMA, derived by conjugation of NHS-DOTA), as the most promising candidate in terms of conjugation rate and yield, stability, antigen immunoreactivity and affinity. Labeling efficiency of the different chelators was investigated with a panel of cold metals indicating DOTAMA as the best chelator. Labeling of Tenatumomab-DOTAMA was then optimized with several metals and stability performed confirms suitability of this conjugate for further development. ST8198AA1 represents an improvement of the previous antibody forms because the labeling with radionuclides like ¹⁷⁷Lu or ⁶⁴Cu would allow theranostic applications in patients bearing tenascin expressing tumors.     

Picomolar affinity antibodies from a fully synthetic naive library selected and evolved by ribosome display

Here we applied ribosome display to in vitro selection and evolution of single-chain antibody fragments (scFvs) from a large synthetic library (Human Combinatorial Antibody Library; HuCAL) against bovine insulin. In three independent ribosome display experiments different clusters of closely related scFvs were selected, all of which bound the antigen with high affinity and specificity. All selected scFvs had affinity-matured up to 40-fold compared to their HuCAL progenitors, by accumulating point mutations during the ribosome display cycles. The dissociation constants of the isolated scFvs were as low as 82 pM, which validates the design of the na?ve library and the power of this evolutionary method. We have thus mimicked the process of antibody generation and affinity maturation with a synthetic library in a cell-free system in just a few days, obtaining molecules with higher affinities than most natural antibodies.     

Selection of functional human antibodies from retroviral display libraries

Antibody library technology represents a powerful tool for the discovery and design of antibodies with high affinity and specificity for their targets. To extend the technique to the expression and selection of antibody libraries in an eukaryotic environment, we provide here a proof of concept that retroviruses can be engineered for the display and selection of variable single-chain fragment (scFv) libraries. A retroviral library displaying the repertoire obtained after a single round of selection of a human synthetic scFv phage display library on laminin was generated. For selection, antigen-bound virus was efficiently recovered by an overlay with cells permissive for infection. This approach allowed more than 10³-fold enrichment of antigen binders in a single selection cycle. After three selection cycles, several scFvs were recovered showing similar laminin-binding activities but improved expression levels in mammalian cells as compared with a laminin-specific scFv selected by the conventional phage display approach. Thus, translational problems that occur when phage-selected antibodies have to be transferred onto mammalian expression systems to exert their therapeutic potential can be avoided by the use of retroviral display libraries.     

Monoclonal antibodies for carcinoembryonic antigen and related antigens as a model system: determination of affinities and specificities of monoclonal antibodies by using biotin-labeled antibodies and avidin as precipitating agent in a solution phase immunoassay

A general method is described for the determination of affinity constants and antigen cross-reactivities of monoclonal antibodies. The method employs biotin-labeled antibody, radiolabeled antigen, and avidin as a precipitating agent in a homogeneous phase, competitive radioimmunoassay. This method eliminates incomplete or variable precipitation of antigen-antibody complexes often encountered in immunoassays in which monoclonal antibodies are employed. Using this assay system, we were able to rapidly determine the affinity constants for a number of monoclonal antibodies elicited to carcinoembryonic antigen (CEA). In the preceding paper it was shown that five of the monoclonal antibodies recognized distinct epitopes on CEA. In antigen-binding experiments with these five monoclonal antibodies, the percent of radiolabeled CEA bound in antibody excess ranged from 30 to 92%. The CEA cross-reacting antigens, normal cross-reacting antigen (NCA), and tumor-extracted, CEA-related antigen (TEX) were significantly bound by one, and to a lesser degree, by two of the five antibodies. Two antibodies did not bind significant amounts of NCA or TEX. In inhibition studies, the amount of unlabeled CEA leading to 50% inhibition of 125I-labeled CEA-binding was in the range of 3.7 to 760 ng per tube. The amount of TEX showing the same degree of inhibition was 23-fold greater than the amount of CEA for two antibodies and 351-fold greater than the amount of CEA for a third antibody. The affinity constants for CEA were in the range of 1.0 x 10(8) to 5.1 x 10(10) M-1. The affinity constants for NCA and TEX, determined for one of the antibodies, were three orders of magnitude lower in comparison to CEA. The heterogeneity of radiolabeled CEA as indicated by the low fraction bound by one of the monoclonal antibodies is shown to be most probably an artifact resulting from radioiodination damage. The application of the approach described in this report should eliminate the problems most commonly encountered in the determination of affinity constants for monoclonal antibodies or the use of monoclonal antibodies in competitive, homogeneous-phase immunoassays.     

Comparative analysis of two immunohistochemical methods for antigen retrieval in the optical lobe of the honeybee Apis mellifera: myosin-V assay

The present study compared two heating methods currently used for antigen retrieval (AR) immunostaining: the microwave oven and the steam cooker. Myosin-V, a molecular motor involved in vesicle transport, was used as a neuronal marker in honeybee Apis mellifera brains fixed in formalin. Overall, the steam cooker showed the most satisfactory AR results. At 100 oC, tissue morphology was maintained and revealed epitope recovery, while evaporation of the AR solution was markedly reduced; this is important for stabilizing the sodium citrate molarity of the AR buffer and reducing background effects. Standardization of heat-mediated AR of formalin-fixed and paraffin-embedded tissue sections results in more reliable immunostaining of the honeybee brain.     

Recombinant human monoclonal antibodies. Basic principles of the immune system transferred to E. coli.

To produce human monoclonal antibodies in bacteria, a gene repertoire of IgM variable regions was isolated from human peripheral B lymphocytes by the polymerase chain reaction. Alternatively, synthetic antibody genes with random hypervariable regions are being generated that may provide libraries of even higher complexity. For the selection of specific monoclonal antibodies from these libraries, we have developed two E. coli vector systems that facilitate the surface display of an antibody physically linked to its own gene. The phagemid pSEX encodes a fusion protein of an antigen binding domain (Fv-antibody) with the docking protein (pIII) of filamentous phages. Specific antibody genes can therefore be enriched by antigen affinity chromatography. The plasmid pAP1 encodes a fusion protein of an Fv-antibody with a bacterial cell-wall protein. Bacteria carrying this plasmid express functional Fv-antibodies tightly bound to their surface. This should enable the selection of single cells with a fluorescence-assisted cell sorter (FACS) using labeled antigen or by adsorption to immobilized antigen. These vectors permit three major principles of the antibody response to be mimicked in E. coli: 1. Generation of a highly complex antibody repertoire; 2. Clonal selection procedures for library screening; and 3. The possibility of increasing a given affinity by repeated rounds of mutation and selection.     

Antibody informatics for drug discovery

More and more antibody therapeutics are being approved every year, mainly due to their high efficacy and antigen selectivity. However, it is still difficult to identify the antigen, and thereby the function, of an antibody if no other information is available. There are obstacles inherent to the antibody science in every project in antibody drug discovery. Recent experimental technologies allow for the rapid generation of large-scale data on antibody sequences, affinity, potency, structures, and biological functions; this should accelerate drug discovery research. Therefore, a robust bioinformatic infrastructure for these large data sets has become necessary. In this article, we first identify and discuss the typical obstacles faced during the antibody drug discovery process. We then summarize the current status of three sub-fields of antibody informatics as follows: (i) recent progress in technologies for antibody rational design using computational approaches to affinity and stability improvement, as well as ab-initio and homology-based antibody modeling; (ii) resources for antibody sequences, structures, and immune epitopes and open drug discovery resources for development of antibody drugs; and (iii) antibody numbering and IMGT. Here, we review “antibody informatics,” which may integrate the above three fields so that bridging the gaps between industrial needs and academic solutions can be accelerated. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.     

Localization of factor VIII-related antigen in the endothelium of the chick embryo chorioallantoic membrane

In this study, by using a polyclonal antibody against factor VIII-related antigen (FVIII-RA), we have examined the expression of FVIII-RA in the blood and lymphatic vessels of the chick embryo chorioallantoic membrane (CAM). The antibody marked the endothelium of blood and lymphatic vessels starting from day 8 of incubation and the cytoplasm of the allantoic epithelial cells. The application of this antibody may be useful for quantifying neovascularization in response to various angiogenic stimuli applied to the CAM. Accepted: 12 October 1999     

Conformational Heterogeneity in Antibody-Protein Antigen Recognition: IMPLICATIONS FOR HIGH AFFINITY PROTEIN COMPLEX FORMATION

Specific, high affinity protein-protein interactions lie at the heart of many essential biological processes, including the recognition of an apparently limitless range of foreign proteins by natural antibodies, which has been exploited to develop therapeutic antibodies. To mediate biological processes, high affinity protein complexes need to form on appropriate, relatively rapid timescales, which presents a challenge for the productive engagement of complexes with large and complex contact surfaces (∼600–1800 Ų). We have obtained comprehensive backbone NMR assignments for two distinct, high affinity antibody fragments (single chain variable and antigen-binding (Fab) fragments), which recognize the structurally diverse cytokines interleukin-1β (IL-1β, β-sheet) and interleukin-6 (IL-6, α-helical). NMR studies have revealed that the hearts of the antigen binding sites in both free anti-IL-1β Fab and anti-IL-6 single chain variable exist in multiple conformations, which interconvert on a timescale comparable with the rates of antibody-antigen complex formation. In addition, we have identified a conserved antigen binding-induced change in the orientation of the two variable domains. The observed conformational heterogeneity and slow dynamics at protein antigen binding sites appears to be a conserved feature of many high affinity protein-protein interfaces structurally characterized by NMR, suggesting an essential role in protein complex formation. We propose that this behavior may reflect a soft capture, protein-protein docking mechanism, facilitating formation of high affinity protein complexes on a timescale consistent with biological processes.     

Engineered pH-dependent recycling antibodies enhance elimination of Staphylococcal enterotoxin B superantigen in mice

A new modality in antibody engineering has emerged in which the antigen affinity is designed to be pH dependent (PHD). In particular, combining high affinity binding at neutral pH with low affinity binding at acidic pH leads to a novel antibody that can more effectively neutralize the target antigen while avoiding antibody-mediated antigen accumulation. Here, we studied how the in vivo pharmacokinetics of the superantigen, Staphylococcal enterotoxin B (SEB), is affected by an engineered antibody with pH-dependent binding. PHD anti-SEB antibodies were engineered by introducing mutations into a high affinity anti-SEB antibody, 3E2, by rational design and directed evolution. Three antibody mutants engineered in the study have an affinity at pH 6.0 that is up to 68-fold weaker than the control antibody. The pH dependency of each mutant, measured as the pH-dependent affinity ratio (PAR – ratio of affinity at pH 7.4 and pH 6.0), ranged from 6.7–11.5 compared to 1.5 for the control antibody. The antibodies were characterized in mice by measuring their effects on the pharmacodynamics and pharmacokinetics (PK) of SEB after co-administration. All antibodies were effective in neutralizing the toxin and reducing the toxin-induced cytokine production. However, engineered PHD antibodies led to significantly faster elimination of the toxin from the circulation than wild type 3E2. The area under the curve computed from the SEB PK profile correlated well with the PAR value of antibody, indicating the importance of fine tuning the pH dependency of binding. These results suggest that a PHD recycling antibody may be useful to treat intoxication from a bacterial toxin by accelerating its clearance.     

Deep mutational scanning of an antibody against epidermal growth factor receptor using mammalian cell display and massively parallel pyrosequencing

We developed a method for deep mutational scanning of antibody complementarity-determining regions (CDRs) that can determine in parallel the effect of every possible single amino acid CDR substitution on antigen binding. The method uses libraries of full length IgGs containing more than 1000 CDR point mutations displayed on mammalian cells, sorted by flow cytometry into subpopulations based on antigen affinity and analyzed by massively parallel pyrosequencing. Higher, lower and neutral affinity mutations are identified by their enrichment or depletion in the FACS subpopulations. We applied this method to a humanized version of the anti-epidermal growth factor receptor antibody cetuximab, generated a near comprehensive data set for 1060 point mutations that recapitulates previously determined structural and mutational data for these CDRs and identified 67 point mutations that increase affinity. The large-scale, comprehensive sequence-function data sets generated by this method should have broad utility for engineering properties such as antibody affinity and specificity and may advance theoretical understanding of antibody-antigen recognition.     

亲和层析法用于噬菌体抗体库的筛选

本研究报道一种基于固定化金属亲和层析(IMAC)的噬菌体抗体库液相筛选方法。将纯化的带有His标签的抗原与噬菌体抗体库混合,噬菌体抗体与抗原充分结合后再加入亲和介质,使噬菌体抗体抗原复合物通过His标签与介质结合,然后通过充分洗涤去除非特异性噬菌体抗体,最后将特异性噬菌体抗体洗脱下来,感染TG1,进行下一轮筛选。整个筛选过程中抗原与抗体的结合在液相中完成,不仅消除了固相介质对抗原表位的影响,也更有利于噬菌体抗体与抗原的充分作用。将此方法应用于HEV NE2蛋白特异性人源噬菌体抗体的筛选,抗原竞争ELISA,阳性血清阻断,可溶性单链抗体表达检测及测序结果表明,最终获得2个特异性人源抗体。