Role of the non‐hypervariable FR3 D‐E loop in single‐domain antibody recognition of haptens and carbohydrates

Single‐domain antibodies (sdAbs), the variable domains of camelid heavy chain‐only antibodies, are generally thought to poorly recognize nonproteinaceous small molecules and carbohydrates in comparison with conventional antibodies. However, the structures of anti‐methotrexate, anti‐triclocarban and anti‐cortisol sdAbs revealed unexpected contributions of the non‐hypervariable “CDR4” loop, formed between β‐strands D and E of framework region 3, in binding. Here, we investigated the potential role of CDR4 in sdAb binding to a hapten, 15‐acetyl‐deoxynivalenol (15‐AcDON), and to carbohydrates. We constructed and panned a phage‐displayed library in which CDR4 of the 15‐AcDON‐specific sdAb, NAT‐267, was extended and randomized. From this library, we identified one sdAb, MA‐232, bearing a 14‐residue insertion in CDR4 and showing improved binding to 15‐AcDON by ELISA and surface plasmon resonance. On the basis of these results, we constructed a second set of phage‐displayed libraries in which the CDR4 and other regions of three hapten‐ or carbohydrate‐binding sdAbs were diversified. With the goal of identifying sdAbs with novel glycan‐binding specificities, we panned the library against four tumor‐associated carbohydrate antigens but were unable to enrich binding phages. Thus, we conclude that while CDR4 may play a role in binding of some rare hapten‐specific sdAbs, diversifying this region through molecular engineering is probably not a general solution to sdAb carbohydrate recognition in the absence of a paired V_L domain.     

Thermal stability of single‐domain antibodies estimated by molecular dynamics simulations

Single‐domain antibodies (sdAbs) function like regular antibodies, however, consist of only one domain. Because of their low molecular weight, sdAbs have advantages with respect to production and delivery to their targets and for applications such as antibody drugs and biosensors. Thus, sdAbs with high thermal stability are required. In this work, we chose seven sdAbs, which have a wide range of melting temperature (T_m) values and known structures. We applied molecular dynamics (MD) simulations to estimate their relative stability and compared them with the experimental data. High‐temperature MD simulations at 400 K and 500 K were executed with simulations at 300 K as a control. The fraction of native atomic contacts, Q, measured for the 400 K simulations showed a fairly good correlation with the T_m values. Interestingly, when the residues were classified by their hydrophobicity and size, the Q values of hydrophilic residues exhibited an even better correlation, suggesting that stabilization is correlated with favorable interactions of hydrophilic residues. Measuring the Q value on a per‐residue level enabled us to identify residues that contribute significantly to the instability and thus demonstrating how our analysis can be used in a mutant case study.     

Evaluation of a noncanonical Cys40‐Cys55 disulfide linkage for stabilization of single‐domain antibodies

Incorporation of noncanonical disulfide linkages into single‐domain antibodies (sdAbs) has been shown to enhance thermostability and other properties. Here, we evaluated the effects of introducing a novel disulfide linkage formed between Cys residues at IMGT positions 40 and 55 on the melting temperatures (T _ms), reversibility of thermal unfolding, solubility, and antigen‐binding affinities of three types of sdAbs (V_HH, V_H, and V_L domains). The Cys40‐Cys55 disulfide linkage was tolerated by 9/9 V_HHs, 12/12 V_Hs, and 2/11 V_Ls tested and its formation was confirmed by mass spectrometry. Using circular dichroism, we found that the Cys40‐Cys55 disulfide linkage increased sdAb T _m by an average of 10.0°C (range: 0–21.8°C). However, enhanced thermostability came at the cost of a partial loss of refolding ability upon thermal denaturation as well as, for some sdAbs, significantly decreased solubility and antigen‐binding affinity. Thus, Cys40/Cys55 can be added to the panel of known locations for introducing stabilizing noncanonical disulfide linkages into antibody variable domains, although its effects should be tested empirically for individual sdAbs.     

Experimental evaluation of single‐domain antibodies predicted by molecular dynamics simulations to have elevated thermal stability

Recently Bekker et al. [Bekker G‐J et al. Protein Sci. 2019;28:429–438.] described a computational strategy of applying molecular‐dynamics simulations to estimate the relative stabilities of single‐domain antibodies, and utilized their method to design changes with the aim of increasing the stability of a single‐domain antibody with a known crystal structure. The structure from which they generated potentially stabilizing mutations is an anti‐cholera toxin single domain antibody selected from a naïve library which has relatively low thermal stability, reflected by a melting point of 48°C. Their work was purely theoretical, so to examine their predictions, we prepared the parental and predicted stabilizing mutant single domain antibodies and examined their thermal stability, ability to refold and affinity. We found that the mutation that improved stability the most (~7°C) was one which changed an amino acid in CDR1 from an asparagine to an aspartic acid. This change unfortunately was also accompanied by a reduction in affinity. Thus, while their modeling did appear to successfully predict stabilizing mutations, introducing mutations in the binding regions is problematic. Of further interest, the mutations selected via their high temperature simulations, did improve refolding, suggesting that they were successful in stabilizing the structure at high temperatures and thereby decrease aggregation. Our result should permit them to reassess and refine their model and may one day lead to a usefulin silico approach to protein stabilization.     

Unique single‐domain antigen binding fragments derived from naturally occurring camel heavy‐chain antibodies

The humoral immune response of camels, dromedaries and llamas includes functional antibodies formed by two heavy chains and no light chains. The amino acid sequence of the variable domain of the naturally occurring heavy‐chain antibodies reveals the necessary adaptations to compensate for the absence of the light chain. In contrast to the conventional antibodies, a large proportion of the heavy‐chain antibodies acts as competitive enzyme inhibitors. Studies on the dromedary immunoglobulin genes start to shed light on the ontogeny of these heavy‐chain antibodies. The presence of the heavy‐chain antibodies and the possibility of immunizing a dromedary allows for the production of antigen binders consisting of a single domain only. These minimal antigen‐binding fragments are well expressed in bacteria, bind the antigen with affinity in the nM range and are very stable. We expect that such camelid single domain antibodies will find their way into a number of biotechnological or medical applications. The structure of the camelid single domain is homologous to the human VH, however, the antigen‐binding loop structures deviate fundamentally from the canonical structures described for human or mouse VHs. This has two additional advantages: (1) the camel or llama derived single domain antibodies might be an ideal scaffold for anti‐idiotypic vaccinations; and (2) the development of smaller peptides or peptide mimetic drugs derived from of the antigen binding loops might be facilitated due to their less complex antigen binding site. Copyright © 1999 John Wiley & Sons, Ltd.     

Biofabrication of antibodies and antigens via IgG‐binding domain engineered with activatable pentatyrosine pro‐tag

We report the assembly of seven different antibodies (and two antigens) into functional supramolecular structures that are specifically designed to facilitate integration into devices using entirely biologically based bottom‐up fabrication. This is enabled by the creation of an engineered IgG‐binding domain (HG3T) with an N‐terminal hexahistidine tag that facilitates purification and a C‐terminal enzyme‐activatable pentatyrosine “pro‐tag” that facilitates covalent coupling to the pH stimuli‐responsive polysaccharide, chitosan. Because we confer pH‐stimuli responsiveness to the IgG‐binding domain, it can be electrodeposited or otherwise assembled into many configurations. Importantly, we demonstrate the loading of both HG3T and antibodies can be achieved in a linear fashion so that quantitative assessment of antibodies and antigens is feasible. Our demonstration formats include: conventional multiwell plates, micropatterned electrodes, and fiber networks. We believe biologically based fabrication (i.e., biofabrication) provides bottom‐up hierarchical assembly of a variety of nanoscale components for applications that range from point‐of‐care diagnostics to smart fabrics. Biotechnol. Bioeng. 2009;103: 231–240. © 2008 Wiley Periodicals, Inc.     

Generation of a phage‐display library of single‐domain camelid VHH antibodies directed against Chlamydomonas reinhardtii antigens,and characterization of VHHs binding cell‐surface antigens

Single‐domain antibodies (sdAbs) are powerful tools for the detection, quantification, purification and subcellular localization of proteins of interest in biological research. We have generated camelid (Lama pacos) heavy chain‐only variable V_H domain (V_HH) libraries against antigens in total cell lysates from Chlamydomonas reinhardtii. The sdAbs in the sera from immunized animals and V_HH antibody domains isolated from the library show specificity to C. reinhardtii and lack of reactivity to antigens from four other algae: Chlorella variabilis, Coccomyxa subellipsoidea, Nannochloropsis oceanica and Thalassiosira pseudonana. Antibodies were produced against a diverse representation of antigens as evidenced by sera ELISA and protein‐blot analyses. A phage‐display library consisting of the V_HH region contained at least 10⁶ individual transformants, and thus should represent a wide range of C. reinhardtii antigens. The utility of the phage library was demonstrated by using live C. reinhardtii cells to pan for V_HH clones with specific recognition of cell‐surface epitopes. The lead candidate V_HH clones (designated B11 and H10) bound to C. reinhardtii with EC₅₀ values ≤0.5 nm . Treatment of cells with V_HH B11 fused to the mCherry or green fluorescent proteins allowed brilliant and specific staining of the C. reinhardtii cell wall and analysis of cell‐wall genesis during cell division. Such high‐complexity V_HH antibody libraries for algae will be valuable tools for algal researchers and biotechnologists.     

The diversity of H3 loops determines the antigen‐binding tendencies of antibody CDR loops

Of the complementarity‐determining regions (CDRs) of antibodies, H3 loops, with varying amino acid sequences and loop lengths, adopt particularly diverse loop conformations. The diversity of H3 conformations produces an array of antigen recognition patterns involving all the CDRs, in which the residue positions actually in contact with the antigen vary considerably. Therefore, for a deeper understanding of antigen recognition, it is necessary to relate the sequence and structural properties of each residue position in each CDR loop to its ability to bind antigens. In this study, we proposed a new method for characterizing the structural features of the CDR loops and obtained the antigen‐binding ability of each residue position in each CDR loop. This analysis led to a simple set of rules for identifying probable antigen‐binding residues. We also found that the diversity of H3 loop lengths and conformations affects the antigen‐binding tendencies of all the CDR loops.     

Surface shapes and surrounding environment analysis of single‐ and double‐stranded DNA‐binding proteins in protein‐DNA interface

Protein‐DNA bindings are critical to many biological processes. However, the structural mechanisms underlying these interactions are not fully understood. Here, we analyzed the residues shape (peak, flat, or valley) and the surrounding environment of double‐stranded DNA‐binding proteins (DSBs) and single‐stranded DNA‐binding proteins (SSBs) in protein‐DNA interfaces. In the results, we found that the interface shapes, hydrogen bonds, and the surrounding environment present significant differences between the two kinds of proteins. Built on the investigation results, we constructed a random forest (RF) classifier to distinguish DSBs and SSBs with satisfying performance. In conclusion, we present a novel methodology to characterize protein interfaces, which will deepen our understanding of the specificity of proteins binding to ssDNA (single‐stranded DNA) or dsDNA (double‐stranded DNA). Proteins 2016; 84:979–989. © 2016 Wiley Periodicals, Inc.     

Induction of the immune response to cell surface antigens in vitro

Summary Two tissue culture incubation systems are described in which immune responses to cell surface antigens have been demonstrated In the one-way “mixed lymphocyte interaction” system, a specific stimulation of thymidine uptake was induced by a particulate membrane antigen fraction, the microsomal lipoproteins (MLP)when low levels (0.01 to 0.001 μg per ml) were incubated with spleen or lymph node cells from nonsensitized mice. No stimulation was seen when allogeneic MLP was used at high levels, 10 μg per ml, nor at any level with syngeneic MLP. Specific effectors were demonstrated after 72-hr incubation with stimulatory levels of allogeneic MLP in three separate in vitro assays, a plaque-forming cell reduction assay, a tumor target assay, and an antigen-binding cell assay. In the latter assay, [¹²⁵I]MLP was used as the source of antigen. This system has limited potential inasmuch as mouse spleen cells do not survive in it beyond the 4th day of culture. The second tissue culture system, the Marbrook system, has much greater possibilities because at least 25% of the inoculum is recovered 7days later. In this culture system a cell-free sheep erythrocyte membrane preparation can induce, plaque-forming cells in the absence of macrophages. Using a sensitive radioimmunoassay, frees specific antibody was detected in culture supernatant fluids. With the same culture system, allogeneic lymphocytotoxic cells (killer) have been induced with spleen cells from unprimed mice in strains differing at the major histocompatibility locus (H-2). Allogeneic MLP induced very significant “killer” cell activity with spleen cells from primed mice. In a syngeneic tumor systems, significant amounts of killer cell activity were induced with unprimed spleen cell inocula, and much larger amounts induced with spleen cells from immunized mice. Presented in the formal symposium on Carcinogenesis in Vitro, at the 25th Annual Meeting of the Tissue Culture Association, Miami Beach, Florida, June 3–6, 1974. This work was supported by Public Health Service Rescarch grants CA 07973 and CA 10815 from the National Cancer Institute.     

Expression of recombinant anti‐colorectal cancer large single‐chain monoclonal antibody in insect cells

Colorectal cancer is the third most commonly diagnosed cancer in the world. Monoclonal antibody (mAb) CO17‐1A recognizes the tumor‐associated antigen GA733‐2, a cell surface glycoprotein highly expressed in colorectal carcinoma cells which is applicable for preventing and curing colorectal cancer. In this study, we tried to produce a new recombinant anti‐colorectal cancer large single chain (lsc) mAb based on mAb CO17‐1A in the baculovirus‐insect cell protein expression system. Two kinds of recombinant lsc mAbs were generated where variable light chain (VL) and heavy chain (HC) of mAb CO17‐1A were fused together by an interchain linker. The only difference between the two mAbs is based on fusion of an ER retention signal (KDEL) at its C‐terminus of HC. Polymerase chain reaction analysis verified the presence of both recombinant genes in the bacmid for generating viral expression vectors in insect cells. Western blot confirmed the expression of lsc mAbs in baculovirus‐infected insect cells. Cell enzyme linked immunosorbent assay (ELISA) showed that the mAbs from cell lysates bound to SW480 and SW620 human colorectal cancer cells. These results indicate that the baculovirus insect expression system can produce anti‐colorectal lsc mAb recognizing human colorectal cancer cells.     

Anchorage‐dependent binding of integrin I‐domain to adhesion ligands

The dynamic interactions between leukocyte integrin receptors and ligands in the vascular endothelium, extracellular matrix, or invading pathogens result in leukocyte adhesion, extravasation, and phagocytosis. This work examined the mechanical strength of the connection between iC3b, a complement component that stimulates phagocytosis, and the ligand‐binding domain, the I‐domain, of integrin α_Mβ₂. Single‐molecule force measurements of α_M I‐domain–iC3b complexes were conducted by atomic force microscope. Strikingly, depending on loading rates, immobilization of the I‐domain via its C‐terminus resulted in a 1.3‐fold to 1.5‐fold increase in unbinding force compared with I‐domains immobilized via the N‐terminus. The force spectra (unbinding force versus loading rate) of the I‐domain–iC3b complexes revealed that the enhanced mechanical strength is due to a 2.4‐fold increase in the lifetime of the I‐domain–iC3b bond. Given the structural and functional similarity of all integrin I‐domains, our result supports the existing allosteric regulatory model by which the ligand binding strength of integrin can be increased rapidly when a force is allowed to stretch the C‐terminus of the I‐domain. This type of mechanism may account for the rapid ligand affinity adjustment during leukocyte migration. Copyright © 2015 John Wiley & Sons, Ltd.     

Redesigning of anti‐c‐Met single chain Fv antibody for the cytoplasmic folding and its structural analysis

Typically, single chain Fv antibodies are unable to fold properly under a reducing cytoplasm because of the reduction of disulfide bonds. The inability to fold limits both the production of the functional scFvs and their targeting against antigens, which are generally executed in a reducing cytoplasm. In this study, the target scFv CDR was grafted with stable human consensus framework sequences, which enabled the generation of a foldable scFv in a reducing cytoplasm of Escherichia coli. Additionally, the structural features affecting the folding efficiency of the engineered scFv were identified by analyzing the predicted structure. An anti‐c‐Met scFv, which was a cytoplasmic non‐foldable protein, was redesigned as the model system. This study confirmed that the engineered anti‐c‐Met scFv was folded into its native form in the cytoplasm of E. coli BL21(DE3) without a significant loss in the specific binding activity against c‐Met antigen. The structures of the wild‐type anti‐c‐Met scFv and the engineered scFv were predicted using homology modeling. A comparative analysis based on the sequence and structure showed that the hydrophobicity of 12 solvent exposed residues decreased, and two newly formed salt bridges might have improved the folding efficiency of the engineered scFv under the reducing condition. Biotechnol. Bioeng. 2010; 106: 367–375. © 2010 Wiley Periodicals, Inc.     

Construction and characterization of two anti‐sweetener single chain antibodies using radioligand binding,fluorescence and circular dichroism spectroscopy

Two single‐chain antibodies (scFv) that bind the superpotent sweetener ligand, NC‐174, were generated from mouse monoclonal antibodies (mAb) NC6.8 (IgG, κ) and NC10.14 (IgG, λ). These scFv were constructed by cloning the variable region sequences of the mAb, connecting them in tandem with a 25‐amino‐acid polypeptide linker, and expressing them in E. coli using the pET‐11a system. The recombinant proteins were purified using Ni²⁺–NTA–agarose by virtue of a hexahistidine sequence introduced to the C‐terminus of the heavy chain variable region during the cloning process. The secondary structure and ligand binding properties of the two scFv, the parent mAbs and proteolytically derived Fab fragments were examined using radioligand binding, circular dichroism (CD) and fluorescence spectroscopy. The far‐UV CD spectra of both scFv possessed predominantly β character, as did those of the Fab, and the near‐UV CD spectral data for scFvNC10.14, NC6.8 and NC10.14 Fab indicated that chromophore perturbation occurred upon ligand binding. The affinity constants determined for the two scFv, Fab and mAb were nearly equivalent. Copyright © 1999 John Wiley & Sons, Ltd.     

Expression of antibodies using single‐open reading frame vector design and polyprotein processing from mammalian cells

We describe a novel polyprotein precursor‐based approach to express antibodies from mammalian cells. Rather than expressing heavy and light chain proteins from separate expression units, the antibody heavy and light chains are contained in one single‐open reading frame (sORF) separated by an intein gene fused in frame. Inside mammalian cells this ORF is transcribed into a single mRNA, and translated into one polypeptide. The antibody heavy and light chains are separated posttranslationally, assembled into the functional antibody molecule, and secreted into culture medium. It is demonstrated that Pol I intein from P. horikoshii mediates protein splicing and cleavage reactions in mammalian cells, in the context of antibody heavy and light chain amino acid sequences. To allow the separation of antibody heavy chain, light chain, and the intein, we investigated a number of intein mutations designed to inhibit intein‐mediated splicing but preserve cleavage reactions. We have also designed constructs in which the signal peptide downstream from intein has altered hydrophobicity. The use of some of these mutant constructs resulted in more efficient antibody secretion, highlighting areas that can be further explored in improving such an expression system. An antibody secreted using one of the sORF constructs was characterized. This antibody has correct N‐terminal sequences for both of its heavy and light chains, correct heavy and light chain MW as well as intact MW as measured by mass spectrometry. Its affinity to antigen, as measured by surface plasmon resonance (SPR), is indistinguishable from that of the same antibody produced using conventional method. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Monoclonal antibodies to sperm surface antigens of the brown alga Fucus serratus exhibit region-, gamete-, species- and genus-preferential binding

A panel of twelve monoclonal antibodies (MAbs), designated FS1 to FS12, have been raised against surface antigens of Fucus serratus sperm. The antibodies were selected on the basis that they show region-, gamete-, species- or genus-preferential binding. Indirect immunofluorescence shows that the antigens bound by the MAbs are distributed non-randomly over the cell surface. Seven MAbs (FS1, FS3, FS4, FS6, FS8, FS9, FS10) bind antigens located primarily on the cell body, while the others (FS2, FS5, FS7, FS11, FS12) bind antigens located primarily on the anterior flagellum. Of the MAbs that label the anterior flagellum, FS2, FS5, FS7 and FS12 form a halo at the perimeter of the flagellum. Electron microscopic-immunogold studies indicate that the halo results from labelling of the mastigonemes, as opposed to the flagellar plasmamembrane. Gamete-preferential binding of antibodies was detected using an enzyme-linked immunosorbent assay with egg membrane vesicles. Eight of the MAbs bind sperm antigens not common to eggs, though FS2, FS4, FS5 and FS9 bind antigens present on both sperm and eggs. In studies of species- and genus-specificity FS2, FS3, FS5, FS6, FS7, FS8, FS10, FS11 and FS12 exhibit genus-preferential binding, labelling sperm of F. serratus and F. vesiculosus more intensely than that of Ascophyllum nodosum. Only FS10 showed marked species-preferential binding, labelling sperm of F. serratus much more intensely than that of F. vesiculosus.Abbreviations Au-GAMIG gold-conjugated goat anti-mouse immunoglobulin - ELISA enzyme-linked immunosorbent assay - EM electron microscope - FITC-RAMIG fluorescein-isothiocyanate-conjugated rabbit anti-mouse immunoglobulin - IIF indirect immunofluorescence - MAb monoclonal antibody     

A novel anti‐prion protein monoclonal antibody and its single‐chain fragment variable derivative with ability to inhibit abnormal prion protein accumulation in cultured cells

mAbs T1 and T2 were established by immunizing PrP gene ablated mice with recombinant MoPrP of residues 121–231. Both mAbs were cross‐reactive with PrP from hamster, sheep, cattle and deer. A linear epitope of mAb T1 was identified at residues 137–143 of MoPrP and buried in PrP^C expressed on the cell surface. mAb T1 showed no inhibitory effect on accumulation of PrP^Sc in cultured scrapie‐infected neuroblastoma (ScN2a) cells. In contrast, mAb T2 recognized a discontinuous epitope ranged on, or structured by, residues 132–217 and this epitope was exposed on the cell surface PrP^C. mAb T2 showed an excellent inhibitory effect on PrP^Sc accumulation in vitro at a 50% inhibitory concentration of 0.02 μg/ml (0.14 nM). The scFv form of mAb T2 (scFv T2) was secreted in neuroblastoma (N2a58) cell cultures by transfection through eukaryotic secretion vector. Coculturing of ScN2a cells with scFv T2‐producing N2a58 cells induced a clear inhibitory effect on PrP^Sc accumulation, suggesting that scFv T2 could potentially be an immunotherapeutic tool for prion diseases by inhibition of PrP^Sc accumulation.