Increasing the potency of neutralizing single-domain antibodies by functionalization with a CD11b/CD18 binding domain

Recombinant single domain antibodies (nanobodies) constitute an attractive alternative for the production of neutralizing therapeutic agents. Their small size warrants rapid bioavailability and fast penetration to sites of toxin uptake, but also rapid renal clearance, which negatively affects their performance. In this work, we present a new strategy to drastically improve the neutralizing potency of single domain antibodies based on their fusion to a second nanobody specific for the complement receptor CD11b/CD18 (Mac-1). These bispecific antibodies retain a small size (˜30 kDa), but acquire effector functions that promote the elimination of the toxin-immunocomplexes. The principle was demonstrated in a mouse model of lethal toxicity with tetanus toxin. Three anti-tetanus toxin nanobodies were selected and characterized in terms of overlapping epitopes and inhibition of toxin binding to neuron gangliosides. Bispecific constructs of the most promising monodomain antibodies were built using anti Mac-1, CD45 and MHC II nanobodies. When co-administered with the toxin, all bispecific antibodies showed higher toxin-neutralizing capacity than the monomeric ones, but only their fusion to the anti-endocytic receptor Mac-1 nanobody allowed the mice to survive a 10-fold lethal dose. In a model of delayed neutralization of the toxin, the anti- Mac-1 bispecific antibodies outperformed a sheep anti-toxin polyclonal IgG that had shown similar neutralization potency in the co-administration experiments. This strategy should have widespread application in the development of nanobody-based neutralizing therapeutics, which can be produced economically and more safely than conventional antisera.     

Pentamerization of single-domain antibodies from phage libraries: a novel strategy for the rapid generation of high-avidity antibody reagents

We describe a novel type of molecule in which single-domain antibodies (sdAbs) isolated from a nai;ve llama single domain antibody library are linked to an oligomerization domain to generate high-avidity, antigen-binding reagents. An sdAb is fused to the B-subunit of Escherichia coli verotoxin, or shiga-like toxin, which self-assembles to form a homopentamer and results in simultaneous sdAb pentamerization and introduction of avidity. Molecular modeling indicated that this fusion protein (PDB: 1OJF), termed pentabody, has structural flexibility for binding to surface-presented antigen. In the instance of an sdAb specific for a peptide antigen, pentamerization resulted in a dramatic increase in functional affinity for immobilized antigen. The pentabody was expressed in high yield in E.coli in a non-aggregated state, and exhibited excellent thermostability and protease resistance. This technology provides a relatively rapid means of generating novel antigen-binding molecules that bind strongly to immobilized antigen. It is expected that pentavalent sdAbs will have general applicability in proteomics, immunochemical staining, cancer diagnosis and other applications in which antigens are presented multivalently.     

Construction of a combinatorial immune library of human single-chain antibodies to orthopoxviruses and selection of antibodies to recombinant prA30L of the variola virus

A combinatorial immune library of human single-chain antibodies (scAbs) was constructed using the genes coding for the variable domains of the heavy and light chains of human immunoglobulins. The genes were cloned from lymphocytes of four subjects vaccinated with the vaccinia virus (VACV). The library included 3 · 10⁷ independent clones. After enrichment with clones producing scAbs against a recombinant analog of the variola virus envelope protein prA30L, the library was used to select a panel of scAbs binding both prA30L and VACV. All scAbs selected were tested for virus-neutralizing activity, and two scAbs proved to suppress VACV plaque formation in monolayers of Vero E6 cells. The specificity of antigen binding was verified by ELISA and Western blotting. The amino acid sequences of the virus-neutralizing scAbs were determined by sequencing their genes.     

Selection of recombinant anti‐SH3 domain antibodies by high‐throughput phage display

Antibodies are indispensable tools in biochemical research and play an expanding role as therapeutics. While hybridoma technology is the dominant method for antibody production, phage display is an emerging technology. Here, we developed and employed a high‐throughput pipeline that enables selection of antibodies against hundreds of antigens in parallel. Binding selections using a phage‐displayed synthetic antigen‐binding fragment (Fab) library against 110 human SH3 domains yielded hundreds of Fabs targeting 58 antigens. Affinity assays demonstrated that representative Fabs bind tightly and specifically to their targets. Furthermore, we developed an efficient affinity maturation strategy adaptable to high‐throughput, which increased affinity dramatically but did not compromise specificity. Finally, we tested Fabs in common cell biology applications and confirmed recognition of the full‐length antigen in immunoprecipitation, immunoblotting and immunofluorescence assays. In summary, we have established a rapid and robust high‐throughput methodology that can be applied to generate highly functional and renewable antibodies targeting protein domains on a proteome‐wide scale.     

Characterization of ICAM-4 binding to the I domains of the CD11a/CD18 and CD11b/CD18 leukocyte integrins.

Intercellular adhesion molecule-4 (ICAM-4, LW blood group antigen), a member of the immunoglobulin superfamily expressed on red cells, has been reported to bind to CD11a/CD18 and CD11b/CD18 leukocyte integrins. The location of the ICAM-4 binding sites on CD11a/CD18 and CD11b/CD18 are not known. CD11/CD18 integrin I domains have been found to act as major binding sites for physiological ligands and a negatively charged glutamic acid in ICAMs is considered important for binding. ICAM-4 lacks such a residue, which is replaced by an arginine. However, we demonstrate here that ICAM-4 in red cells and transfected fibroblasts interacts specifically with the I domains of CD11a/CD18 and CD11b/CD18 integrins. The binding was inhibited by anti-I domain and anti-ICAM-4 antibodies and it was dependent on divalent cations. Interestingly, ICAM-4 negative red cells were still able to bind to the CD11b/CD18 I domain but the binding of these cells to the CD11a/CD18 I domain was clearly reduced. Using a solid phase assay, we were able to show that isolated I domains directly and specifically bind to purified recombinant ICAM-4 in a cation dependent manner. Competition experiments indicated that the binding sites in ICAM-4 for the CD11a and CD11b I domains are different. However, the ICAM-4 binding region in both I domains seems to overlap with the regions recognized by the ICAM-1 and ICAM-2. Thus we have established that the I domains contain an ICAM-4 binding region in CD11a/CD18 and CD11b/CD18 leukocyte integrins.     

Identification of Bacillus thuringiensis Cry3Aa toxin domain II loop 1 as the binding site of Tenebrio molitor cadherin repeat CR12

Bacillus thuringiensis Cry toxins exert their toxic effect by specific recognition of larval midgut proteins leading to oligomerization of the toxin, membrane insertion and pore formation. The exposed domain II loop regions of Cry toxins have been shown to be involved in receptor binding. Insect cadherins have shown to be functionally involved in toxin binding facilitating toxin oligomerization. Here, we isolated a VHH (VHHA5) antibody by phage display that binds Cry3Aa loop 1 and competed with the binding of Cry3Aa to Tenebrio molitor brush border membranes. VHHA5 also competed with the binding of Cry3Aa to a cadherin fragment (CR12) that was previously shown to be involved in binding and toxicity of Cry3Aa, indicating that Cry3Aa binds CR12 through domain II loop 1. Moreover, we show that a loop 1 mutant, previously characterized to have increased toxicity to T. molitor, displayed a correlative enhanced binding affinity to T. molitor CR12 and to VHHA5. These results show that Cry3Aa domain II loop 1 is a binding site of CR12 T. molitor cadherin.     

Two leukocyte receptors (CD11a/CD18 and CD11b/CD18) mediate transient adhesion to endothelium by binding to different ligands

Upon stimulation with C5a, TNF, or phorbol dibutyrate (PDB), polymorphonuclear leukocytes (PMN) exhibit first an increase then a decrease in adhesion to unstimulated endothelial cells (EC). Essentially all of this adhesion is mediated by the CD18 family of leukocyte integrins on PMN. To determine the individual roles of CD11a/CD18 (LFA-1), CD11b/CD18 (CR3, Mac-1) and CD11c/CD18 (p150,95) in adhesion of PDB-stimulated PMN to unstimulated EC, mAb against the CD11 chains were used. mAb against CD11a or CD11b each blocked adhesion of PMN to EC by approximately 50%, but mAb against CD11c had no effect. Inasmuch as a combination of anti-CD11a and CD11b mAb completely blocked adhesion, it appears that CD11a/CD18 and CD11b/CD18 make approximately equal contributions to binding, and CD11c does not participate. Anti-CD11a or CD11b each blocked adhesion by about 50% throughout the transient time course of PDB-stimulated adhesion, indicating that the capacity of each of these receptors to bind EC is transiently activated by PDB. We next examined the role of ICAM-1 on EC as a ligand for CD18. Two anti-ICAM-1 mAb (LB-2 and 84H10) each inhibited PMN adhesion in a dose-dependent fashion, reaching a maximal inhibition of approximately 50%. Anti-ICAM-1 mAb blocked the CD11a/CD18-dependent portion of adhesion because concomitant use of anti-CD11a and anti-ICAM-1 did not cause additive inhibition. In contrast, anti-CD11b plus anti-ICAM-1 resulted in complete blockade of adhesion. This result suggests that CD11a/CD18 recognizes ICAM-1 on EC, but CD11b/CD18 recognizes a different ligand(s). To determine if CD11b CD18 has the ability to recognize ICAM-1, human macrophages were plated on culture surfaces coated with purified ICAM-1. Interaction of CD11a/CD18 with the surface-bound ICAM-1 resulted in selective down-modulation of CD11a/CD18 from the apical portion of the macrophages. In contrast, ICAM-1-coated surfaces did not down-modulate CD11b/CD18. The data suggest that CD11b/CD18 does not recognize ICAM-1, and that this receptor functions in adhesion of PMN to EC by recognizing novel ligand(s) on EC.     

IgE immunotherapy: A novel concept with promise for the treatment of cancer

The importance of antibodies in activating immune responses against tumors is now better appreciated with the emergence of checkpoint blockade antibodies and with engineered antibody Fc domains featuring enhanced capacity to focus potent effector cells against cancer cells. Antibodies designed with Fc regions of the IgE class can confer natural, potent, long-lived immune surveillance in tissues through tenacious engagement of high-affinity cognate Fc receptors on distinct, often tumor-resident immune effector cells, and through ability to activate these cells under tumor-induced Th2-biased conditions. Here, we review the properties that make IgE a contributor to the allergic response and a critical player in the protection against parasites, which also support IgE as a novel anti-cancer modality. We discuss IgE-based active and passive immunotherapeutic approaches in disparate in vitro and in vivo model systems, collectively suggesting the potential of IgE immunotherapies in oncology. Translation toward clinical application is now in progress.     

Polymorphonuclear granulocyte expression of CD11a/CD18, CD11b/CD18 and L-selectin in normal individuals

Abstract In this study direct immunofluorescence and flow cytometry with calibration using quantitative bead standards were used to enumerate the cell surface receptors CD11a/CD18, CD11b/CD18 and L-selectin. Holding blood at room temperature and fixation of samples prior to staining induced changes in expression, while immediate staining of polymorphonuclear granulocytes (PMN) in whole blood followed by fixation produced accurate values. The ranges of PMN adhesion molecule expression in 10 normal individuals were CD11a/CD18: 14794–28725, CD11b/CD18: 5300–11939 and L-selectin: 35662–61654 receptors per cell. Differences within individuals over 4 h were also observed. Adhesion molecule expression is used as an index of the adhesive function and state of activation of the cell. The data presented here shows that there is inherent variability in the expression of the PMN adhesion molecules between and within individuals, thus direct comparisons of PMN adhesion molecule expression between patients and “normals” must be interpreted with caution.     

Inhibition of HER3 activation and tumor growth with a human antibody binding to a conserved epitope formed by domain III and IV

Human epidermal growth factor receptor 3 (HER3, also known as ErbB3) has emerged as relevant target for antibody-mediated tumor therapy. Here, we describe a novel human antibody, IgG 3–43, recognizing a unique epitope formed by domain III and parts of domain IV of the extracellular region of HER3, conserved between HER3 and mouse ErbB3. An affinity of 11 nM was determined for the monovalent interaction. In the IgG format, the antibody bound recombinant bivalent HER3 with subnanomolar affinity (K_D = 220 pM) and HER3-expressing tumor cells with EC₅₀ values in the low picomolar range (27 - 83 pM). The antibody competed with binding of heregulin to HER3-expressing cells, efficiently inhibited phosphorylation of HER3 as well as downstream signaling, and induced receptor internalization and degradation. Furthermore, IgG 3–43 inhibited heregulin-dependent proliferation of several HER3-positive cancer cell lines and heregulin-independent colony formation of HER2-overexpressing tumor cell lines. Importantly, inhibition of tumor growth and prolonged survival was demonstrated in a FaDu xenograft tumor model in SCID mice. These findings demonstrate that by binding to the membrane-proximal domains III and IV involved in ligand binding and receptor dimerization, IgG 3–43 efficiently inhibits activation of HER3, thereby blocking tumor cell growth both in vitro and in vivo.     

应用噬菌体随机肽库技术筛选戊型肝炎病毒中和表位模拟肽

以识别戊型肝炎病毒（HEV）构象依赖性中和表位的单克隆抗体8C11、8H3作为固相筛选分子,对噬菌体随机7肽库进行4轮筛选后,随机挑取单克隆噬菌体进行测序。合成优势7肽序列基因,将其插入HBcAg-AA78-83位置之中,进行原核表达,所获重组蛋白经蛋白印迹实验证实可与相应单抗结合,电镜下可见重组蛋白能形成与HBcAg相似的类病毒颗粒。化学合成单抗8H3筛选出的优势7肽,所获7肽经生物传感器结合实验证实与单抗8H3结合。这些结果提示用噬菌体7肽库可以筛选出部分模拟构象性表位的短肽,为亚单位疫苗的研制提供了新的思路。     

Purification of anti-MUC1 antibodies by peptide mimotope affinity chromatography using peptides derived from a polyvalent phage display library

A polyvalent, lytic phage display system (T7Select415-1b) displaying a random peptide library has been investigated for its ability to discover novel mimotopes reactive with the therapeutic monoclonal antibody C595. Sequence analysis of enriched phage lead to the identification of a predominant sequence RNREAPRGKICS, and two other consensus sequences RXXP and RXP. The novel synthetic peptide RNREAPRGKICS was linked to beaded agarose and the performance as a mimotope affinity chromatography matrix evaluated. Antibody purified using the novel matrix was found to be of higher specific reactivity than antibody purified using the conventional epitope matrix (peptide APDTRPAPG). The RNREAPRGKICS peptide binding to C595 demonstrated a higher equilibrium association constant (K(A)=0.75 x 10(6)) than the epitope peptide (K(A)=0.16 x 10(6)). Circular dichroism showed that the novel peptide had a more highly ordered structure at 4 degrees C and room temperature, than the epitope peptide.     

Evolution of binding affinity in a WW domain probed by phage display

The WW domain is an approximately 38 residue peptide-binding motif that binds a variety of sequences, including the consensus sequence xPPxY. We have displayed hYAP65 WW on the surface of M13 phage and randomized one-third of its three-stranded antiparallel beta-sheet. Improved binding to the hydrophobic peptide, GTPPPPYTVG (WW1), was selected in the presence of three different concentrations of proteinase K to simultaneously drive selection for improved stability as well as high-affinity binding. While some of the selected binders show cooperative unfolding transitions, others show noncooperative thermal unfolding curves. Two novel WW consensus sequences have been identified, which bind to the xPPxY motif with higher affinity than the wild-type hYAP65 WW domain. These WW domain sequences are not precedented in any natural WW domain sequence. Thus, there appear to be a large number of motifs capable of recognizing the target peptide sequence, only a subset of which appear to be used in natural proteins.     

The remarkable flexibility of the human antibody repertoire; isolation of over one thousand different antibodies to a single protein, BLyS

It is well established that the humoral immune response can generate antibodies to many different antigens. The antibody diversity required to achieve this is believed to be substantial. However, the extent to which the immune repertoire can generate structural diversity against a single target antigen has never been addressed. Here, we have used phage display to demonstrate the extraordinary capacity of the human antibody repertoire. Over 1000 antibodies, all different in amino acid sequence, were generated to a single protein, B-lymphocyte stimulator (BLyS™ protein). This is a highly diverse panel of antibodies as exemplified by the extensive heavy and light chain germline usage: 42/49 functional heavy chain germlines and 19/33 V_λ and 13/35 V_κ light chain germlines were all represented in the panel of antibodies. Moreover, a high level of sequence diversity was observed in the V_H CDR3 domains of these antibodies, with 568 different amino acid sequences identified. Thus we have demonstrated that specific recognition of a single antigen can be achieved from many different VDJ combinations, illustrating the remarkable problem-solving ability of the human immune repertoire. When studied in a biochemical assay, around 500 (40%) of these antibodies inhibited the binding of BLyS to its receptors on B-cell lines. The most potent antibodies inhibited BLyS binding with sub-nanomolar IC₅₀ values and with sub-nanomolar affinities. Such antibodies provide excellent choices as candidates for the treatment of BLyS-associated autoimmune diseases.     

High-throughput screening based identification of small molecule antagonists of integrin CD11b/CD18 ligand binding

Binding of leukocyte specific integrin CD11b/CD18 to its physiologic ligands is important for the development of normal immune response in vivo. Integrin CD11b/CD18 is also a key cellular effector of various inflammatory and autoimmune diseases. However, small molecules selectively inhibiting the function of integrin CD11b/CD18 are currently lacking. We used a newly described cell-based high-throughput screening assay to identify a number of highly potent antagonists of integrin CD11b/CD18 from chemical libraries containing >100,000 unique compounds. Computational analyses suggest that the identified compounds cluster into several different chemical classes. A number of the newly identified compounds blocked adhesion of wild-type mouse neutrophils to CD11b/CD18 ligand fibrinogen. Mapping the most active compounds against chemical fingerprints of known antagonists of related integrin CD11a/CD18 shows little structural similarity, suggesting that the newly identified compounds are novel and unique.     

Methodologies for the isolation of alternative binders with improved clinical potentiality over conventional antibodies

The availability of binders to different functional domains of the same protein or to physiologically co-operating proteins allows for the simultaneous inhibition of independent downstream signaling pathways. This multi-target approach represents a promising therapeutic strategy, as demonstrated in the case of the synergistic effect of anti-Her2 treatment based on the combined use of the trastuzumab and pertuzumab monoclonal antibodies that induce cellular cytotoxicity and impair the receptor dimerization, respectively. Therefore, a reliable selection method for the recovery of epitope-specific antibodies is highly needed. Animal immunization with short peptides resembling the epitope sequence for raising conventional antibodies represents an alternative. Panning phage displayed libraries of recombinant antibodies such as scFvs and nanobodies or of other peptide collections is another option. Although recombinant antibodies can provide the same specificity as conventional antibodies, they offer at least two further advantages: i) the protocols for the selection of epitope-specific antibodies can be rationally designed, and ii) their expression as multivalent, bispecific and biparatopic molecules is feasible. This review will analyze the recent literature concerning technical aspects related to the isolation, the expression as multivalent molecules, and the therapeutic applications of binders able to interfere with antigen functional domains. The term binder will be preferred when possible to include those molecules, such as peptides or affibodies, with at least some proven practical uses.     

CD11a/CD18 and CD11b/CD18 modulation by lipoteichoic acid, N-acetyl-muramyl-α-alanyl-d-isoglutamine, muramic acid and protein A from Staphylococcus aureus

Abstract This study investigates the effect of some components of the Staphylococcus aureus cell wall [lipoteichoic acid (LTA), N -acetyl-muramyl-alanyl- d -isoglutamine (MD), muramic acid (MA) and protein A (PA)] in modulating expression of cell-surface adhesion molecules CD11a/CD18, CD11b/CD18 on monocytes qualitatively and quantitatively. Monocytes incubated with bacterial components presented different CD11b/CD18 expressions which were dose-dependent in contrast to controls. The results obtained demonstrated that lymphocytes incubated with bacterial components also increased the expression of CD11a/CD18. The modifications in activation of CD11a/CD18 and CD11b/CD18 expression are probably correlated with modifications of membrane fluidity measured as polarisation fluorescence (P).     

CD11b/CD18-dependent interactions of neutrophils with intestinal epithelium are mediated by fucosylated proteoglycans

CD11b/CD18-mediated adhesive interactions play a key role in regulating polymorphonuclear leukocytes (PMN)) migration across intestinal epithelium. However, the identity of epithelial ligands for migrating PMN remains obscure. In this study we investigated the role of carbohydrates in mediating adhesive interactions between T84 intestinal epithelial cells and CD11b/CD18 purified from PMN. Fucoidin, heparin/heparin sulfate, N-acetyl-D-glucosamine, mannose-6-phosphate, and laminarin were found to inhibit adhesion of T84 cells to CD11b/CD18. The most potent inhibitory effects were observed with fucoidin (50% inhibition at 1-5 x 10(-8) M). Binding assays demonstrated that fucoidin directly bound to CD11b/CD18 in a divalent cation- and sulfation-dependent fashion that was blocked by anti-CD11b mAbs. Experiments employing CD11b/CD18 as a probe to blot T84 cell fucosylated proteins purified via fucose-specific lectin column revealed several candidate CD11b/CD18 binding proteins with molecular masses of 95, 50, 30, 25, and 20 kDa. Fucosidase treatment of T84 cells resulted in significantly reduced cell adhesion to CD11b/CD18, while no inhibition was observed after neuraminidase treatment. Finally, significant inhibition of T84 cell adhesion to CD11b/CD18 was observed after blocking cell proteoglycan synthesis with p-nitrophenyl-beta-D-xylopyranoside. These findings implicate epithelial cell surface proteoglycans decorated with sulfated fucose moieties as ligands for CD11b/CD18 during PMN migration across mucosal surfaces.     

CD157 is part of a supramolecular complex with CD11b/CD18 on the human neutrophil cell surface

CD157 is a GPI-anchored cell surface glycoprotein expressed by human peripheral blood neutrophils. Cross-linking of CD157 induces intracellular Ca2+ mobilization and re-shaping in neutrophils, thus regulating their adhesive and migratory properties. Results obtained by immunolocalization and confocal microscopy indicate that CD157 lies in close proximity to the CD11b/CD18 complex which is strongly expressed on the activated neutrophil cell membrane where it plays a predominant role in adhesion. This study analyses the physical association between CD157 and CD18 in human neutrophils by co-immunoprecipitation experiments. The anti-CD157 monoclonal antibody RF3 co-precipitates CD18, and the anti-CD18 antibody TS1/18 co-precipitates CD157 from human neutrophil lysates. These results confirm that CD157 physically interacts with CD11b/CD18 complex in human neutrophils.     

Function of the lectin domain of Mac-1/complement receptor type 3 (CD11b/CD18) in regulating neutrophil adhesion

A lectin function within CD11b mediates both cytotoxic priming of Mac-1/complement receptor type 3 (CR3) by beta-glucan and the formation of transmembrane signaling complexes with GPI-anchored glycoproteins such as CD16b (FcgammaRIIIb). A requirement for GPI-anchored urokinase plasminogen activator receptor (uPAR; CD87) in neutrophil adhesion and diapedesis has been demonstrated with uPAR-knockout mice. In this study, neutrophil activation conditions generating high-affinity (H-AFN) or low-affinity (L-AFN) beta(2) integrin adhesion were explored. A role for the Mac-1/CR3 lectin domain and uPAR in mediating H-AFN or L-AFN adhesion was suggested by the inhibition of Mac-1/CR3-dependent adhesion to ICAM-1 or fibrinogen by beta-glucan or anti-uPAR. The formation of uPAR complexes with Mac-1/CR3 activated for L-AFN adhesion was demonstrated by fluorescence resonance energy transfer. Conversely, Jurkat cell LFA-1 H-AFN-adhesion to ICAM-1 was not associated with uPAR/LFA-1 complexes, any requirement for GPI-anchored glycoproteins, or inhibition by beta-glucan. A single CD11b lectin site for beta-glucan and uPAR was suggested because the binding of either beta-glucan or uPAR to Mac-1/CR3 selectively masked two CD11b epitopes adjacent to the transmembrane domain. Moreover, treatment with phosphatidylinositol-specific phospholipase C that removed GPI-anchored proteins increased CD11b-specific binding of (125)I-labeled beta-glucan by 3-fold and this was reversed with soluble recombinant uPAR. Conversely, neutrophil activation for generation of Mac-1/CR3/uPAR complexes inhibited CD11b-dependent binding of (125)I-labeled beta-glucan by 75%. These data indicate that the same lectin domain within CD11b regulates both the cytotoxic and adhesion functions of Mac-1/CR3.