Guinea pig peritoneal macrophages. Differential effects of lectins on interaction with IgG immunoglobulins

Guinea pig peritoneal macrophages have on their surface two receptors, one (Fcγ₁/γ₂R) binding both guinea pig IgG1 and IgG2 and the second (Fcγ₂R) binding only IgG2 immunoglobulins. We have previously shown that treatment of macrophages with neuraminidase or glycosylation inhibitors affects, in a different way, the binding of guinea pig IgG1, IgG2, and rabbit IgG. In the present study we have shown that pretreatment of guinea pig macrophages with lectins (Con A, WGA, and PNA) also has a different effect on the interaction of the cells with IgG. The lectins increased the binding of guinea pig IgG1, whereas rabbit IgG and guinea pig IgG2 were bound with a lower efficiency than in the case of control cells. Since sialic acid residues seem to modulate the activity of receptors and WGA interacts with sialylated oligosaccharides, we determined the IgG-binding characteristics for WGA-pretreated macrophages. We found that the increase in IgG1-binding ability was caused by an increase in the value of K_app, but the number of IgG-binding sites was lower than in the control cells. In the case of rabbit IgG and guinea pig IgG2 we observed a decrease of both the value of K_app and the number of IgG-binding sites. WGA did not interact directly with the Fcγ receptor. The results of our former papers and the different effects of lectins of various specificities described in this paper suggest different positions of Fcγ₁/γ₂ and Fcγ₂R in the plane of the plane of the macrophage membrane in respect to various membrane glycoconjugates. Interaction of IgG with macrophage Fcγ receptors depends in a different way on glycoconjugates on the surface of the macrophage. Our results suggest that changes in glycosylation of macrophage surface glycoconjugates may be used by the cell for regulating the binding activities of the macrophage Fcγ receptors.     

An antibody Fc engineered for conditional antibody-dependent cellular cytotoxicity at the low tumor microenvironment pH

Despite the exquisite specificity and high affinity of antibody-based cancer therapies, treatment side effects can occur since the tumor-associated antigens targeted are also present on healthy cells. However, the low pH of the tumor microenvironment provides an opportunity to develop conditionally active antibodies with enhanced tumor specificity. Here, we engineered the human IgG1 Fc domain to enhance pH-selective binding to the receptor FcγRIIIa and subsequent antibody-dependent cellular cytotoxicity (ADCC). We displayed the Fc domain on the surface of mammalian cells and generated a site-directed library by altering Fc residues at the Fc–FcγRIIIa interface to support interactions with positively charged histidine residues. We then used a competitive staining and flow cytometric selection strategy to isolate Fc variants exhibiting reduced FcγRIIIa affinities at neutral pH, but physiological affinities at the tumor-typical pH 6.5. We demonstrate that antibodies composed of Fab arms binding the breast cell epithelial marker Her2 and the lead Fc variant, termed acid-Fc, exhibited an ∼2-fold pH-selectivity for FcγRIIIa binding based on the ratio of equilibrium dissociation constants K_d,7.4/K_d,6.5, due to a faster dissociation rate at pH 7.4. Finally, in vitro ADCC assays with human FcγRIIIa-positive natural killer and Her2-positive target cells demonstrated similar activities for anti-Her2 antibodies bearing the wild-type or acid-Fc at pH 6.5, but nearly 20-fold reduced ADCC for acid-Fc at pH 7.4, based on EC₅₀ ratios. This work shows the promise of mammalian cell display for Fc engineering and the feasibility of pH-selective Fc activation to provide a second dimension of selective tumor cell targeting.     

Constant domain-exchanged Fab enables specific light chain pairing in heterodimeric bispecific SEED-antibodies

BackgroundBispecific antibodies promise to broadly expand the clinical utility of monoclonal antibody technology. Several approaches for heterodimerization of heavy chains have been established to produce antibodies with two different Fab arms, but promiscuous pairing of heavy and light chains remains a challenge for their manufacturing.MethodsWe have designed a solution in which the C_H1 and C_L domain pair in one of the Fab fragments is replaced with a C_H3-domain pair and heterodimerized to facilitate correct modified Fab-chain pairing in bispecific heterodimeric antibodies based on a strand-exchange engineered domain (SEED) scaffold with specificity for epithelial growth factor receptor and either CD3 or CD16 (FcγRIII).ResultsBispecific antibodies retained binding to their target antigens and redirected primary T cells or NK cells to induce potent killing of target cells. All antibodies were expressed at a high yield in Expi293F cells, were detected as single sharp symmetrical peaks in size exclusion chromatography and retained high thermostability. Mass spectrometric analysis revealed specific heavy-to-light chain pairing for the bispecific SEED antibodies as well as for one-armed SEED antibodies co-expressed with two different competing light chains.ConclusionIncorporation of a constant domain-exchanged Fab fragment into a SEED antibody yields functional molecules with favorable biophysical properties.General significanceOur results show that the novel engineered bispecific SEED antibody scaffold with an incorporated Fab fragment with C_H3-exchanged constant domains is a promising tool for the generation of complete heterodimeric bispecific antibodies with correct light chain pairing.     

Construction of a novel synergistic system for production and recovery of secreted recombinant proteins by the cell surface engineering

We determined whether the cocultivation of yeast cells displaying a ZZ-domain and secreting an Fc fusion protein can be a novel tool for the recovery of secreted recombinant proteins. The ZZ-domain from Staphylococcus aureus protein A was displayed on the cell surface of Saccharomyces cerevisiae under the control of the GAL1 promoter. Strain S. cerevisiae BY4742 cells displaying the ZZ-domain on their surface were used for cocultivation with cells that produce a target protein fused to the Fc fragment as an affinity tag. The enhanced green fluorescent protein or Rhizopus oryzae lipase was genetically fused to the N and C termini of the Fc fragment of human immunoglobulin G, respectively. Through analysis by fluorescence-activated cell sorting and enzymatic assay, it was demonstrated that these fusion proteins are successfully produced in the medium and recovered by affinity binding with the cell surface displaying the ZZ-domain. These results suggest that the ZZ-domain-displaying cell and Fc fusion protein-secreting cell can be applied to use in synergistic process of production and recovery of secreted recombinant proteins.     

Biotinylation of the Fcγ receptor ectodomains by mammalian cell co‐transfection: application to the development of a surface plasmon resonance‐based assay

We here report the production of four biotinylated Fcγ receptor (FcγR) ectodomains and their subsequent stable capture on streptavidin‐biosensor surfaces. For receptor biotinylation, we first describe an in‐cell protocol based on the co‐transfection of two plasmids corresponding to one of the FcγR ectodomains and the BirA enzyme in mammalian cells. This strategy is compared with a standard sequential in vitro enzymatic biotinylation with respect to biotinylation level and yield. Biotinylated FcγR ectodomains that have been prepared with both strategies are then compared by analytical ultracentrifugation and surface plasmon resonance (SPR) analyses. Overall, we demonstrate that in‐cell biotinylation is an interesting alternative to standard biotinylation protocol, as it requires less purification steps while yielding higher titers. Finally, biotin‐tagged FcγRs produced with the in‐cell approach are successfully applied to the development of SPR‐based assays to evaluate the impact of the glycosylation pattern of monoclonal antibodies on their interaction with CD16a and CD64. In that endeavor, we unambiguously observe that highly galactosylated trastuzumab (TZM‐gal), non‐glycosylated trastuzumab (TZM‐NG), and reference trastuzumab are characterized by different kinetic profiles upon binding to CD16a and CD64 that had been captured at the biosensor surface via their biotin tag. More precisely, while TZM‐NG binding to CD16a was not detected, TZM‐gal formed a more stable complex with CD16a than our reference TZM. In contrast, both glycosylated TZM bound to captured CD64 in a stable and similar fashion, whereas the interaction of their non‐glycosylated form with CD64 was characterized by a higher dissociation rate. Copyright © 2015 John Wiley & Sons, Ltd.     

High-efficiency recovery of target cells using improved yeast display system for detection of protein–protein interactions

We constructed a high-throughput screening (HTS) system for target cells based on the detection of protein–protein interactions by flow cytometric sorting due to the improvement in the yeast cell surface display system. Interaction model proteins, which are the ZZ domain derived from Staphylococcus aureus and the Fc part of human immunoglobulin G (IgG), were displayed on the yeast cell surface. We achieved a rapid and enhanced expression of these proteins as a result of adopting an appropriate yeast strain and a suitable promoter. The displayed ZZ domain had an ability to bind to rabbit IgG and the displayed Fc part to protein A. These were confirmed by flow cytometry and fluorescence microscopy. Furthermore, the cells displaying the ZZ domain or Fc part were isolated from the model libraries constructed by mixing the control yeast cells with the target yeast cells. The ratio of the target cells was increased from 0.0001% to more than 70% by two cycles of cell sorting. These results indicate that we can achieve a rapid and highly efficient isolation method for the target cells with FACSCalibur and that this method will further extend the application of flow cytometric sorting to library selections.     

pH dependence and stoichiometry of binding to the Fc region of IgG by the herpes simplex virus Fc receptor gE-gI

Herpes simplex virus type 1 encodes two glycoproteins, gE and gI, that form a heterodimer on the surface of virions and infected cells. The gE-gI heterodimer has been implicated in cell-to-cell spread of virus and is a receptor for the Fc fragment of IgG. Previous studies localized the gE-gI-binding site on human IgG to a region near the interface between the C(H)2 and C(H)3 domains of Fc, which also serves as the binding site for bacterial and mammalian Fc receptors. Although there are two potential gE-gI-binding sites per Fc homodimer, only one gE-gI heterodimer binds per IgG in gel filtration experiments. Here we report production of recombinant human Fc molecules that contain zero, one, or two potential gE-gI-binding sites and use them in analytical ultracentrifugation experiments to show that two gE-gI heterodimers can bind to each Fc. Further characterization of the gE-gI interaction with Fc reveals a sharp pH dependence of binding, with K(D) values of approximately 340 and approximately 930 nm for the first and second binding events, respectively, at the slightly basic pH of the cell surface (pH 7.4), but undetectable binding at pH 6.0. This strongly pH-dependent interaction suggests a physiological role for gE-gI dissociation from IgG within acidic intracellular compartments, consistent with a mechanism whereby herpes simplex virus promotes intracellular degradation of anti-viral antibodies.     

Single-step Protein A and Protein G avidity purification methods to support bispecific antibody discovery and development

ABSTRACT

Heavy chain (Hc) heterodimers represent a majority of bispecific antibodies (bsAbs) under clinical development. Although recent technologies achieve high levels of Hc heterodimerization (HD), traces of homodimer contaminants are often present, and as a consequence robust purification techniques for generating highly pure heterodimers in a single step are needed. Here, we describe two different purification methods that exploit differences in Protein A (PA) or Protein G (PG) avidity between homo- and heterodimers. Differential elution between species was enabled by removing PA or PG binding in one of the Hcs of the bsAb. The PA method allowed the avidity purification of heterodimers based on the VH3 subclass, which naturally binds PA and interferes with separation, by using a combination of IgG3 Fc and a single amino acid change in VH3, N82aS. The PG method relied on a combination of three mutations that completely disrupts PG binding, M428G/N434A in IgG1 Fc and K213V in IgG1 CH1. Both methods achieved a high level of heterodimer purity as single-step techniques without Hc HD (93–98%). Since PA and PG have overlapping binding sites with the neonatal Fc receptor (FcRn), we investigated the effects of our engineering both in vitro and in vivo. Mild to moderate differences in FcRn binding and Fc thermal stability were observed, but these did not significantly change the serum half-lives of engineered control antibodies and heterodimers. The methods are conceptually compatible with various Hc HD platforms such as BEAT® (Bispecific Engagement by Antibodies based on the T cell receptor), in which the PA method has already been successfully implemented.     

Killer to cure: Expression and production costs calculation of tobacco plant-made cancer-immune checkpoint inhibitors

Immune checkpoint inhibitors (ICIs) have achieved huge clinical success. However, many still have limited response rates, and are prohibitively costly. There is a need for effective and affordable ICIs, as well as local manufacturing capacity to improve accessibility, especially to low-to-middle income countries (LMICs). Here, we have successfully expressed three key ICIs (anti-PD-1 Nivolumab, anti-NKG2A Monalizumab, and anti-LAG-3 Relatimab) transiently in Nicotiana benthamiana and Nicotiana tabacum plants. The ICIs were expressed with a combination of different Fc regions and glycosylation profiles. They were characterized in terms of protein accumulation levels, target cell binding, binding to human neonatal Fc receptors (hFcRn), human complement component C1q (hC1q) and various Fcγ receptors, as well as protein recovery during purification at 100 mg- and kg-scale. It was found that all ICIs bound to the expected target cells. Furthermore, the recovery during purification, as well as Fcγ receptor binding, can be altered depending on the Fc region used and the glycosylation profiles. This opens the possibility of using these two parameters to fine-tune the ICIs for desired effector functions. A scenario-based production cost model was also generated based on two production scenarios in hypothetical high- and low-income countries. We have shown that the product accumulation and recovery of plant production platforms were as competitive as mammalian cell-based platforms. This highlights the potential of plants to deliver ICIs that are more affordable and accessible to a widespread market, including LMICs.     

Influence of the bispecific antibody IgG subclass on T cell redirection

ABSTRACT

T cell redirection mediated by bispecific antibodies (BsAbs) is a promising cancer therapy. Dual antigen binding is necessary for potent T cell redirection and is influenced by the structural characteristics of a BsAb, which are dependent on its IgG subclass. In this study, model BsAbs targeting CD19xCD3 were generated in variants of IgG1, IgG2, and IgG4 carrying Fc mutations that reduce FcγR interaction, and two chimeric IgG subclasses termed IgG1:2 and IgG4:2, in which the IgG1- or IgG4-F(ab)₂ are grafted on an IgG2 Fc. Molecules containing an IgG2 or IgG4-F(ab)₂ domain were confirmed to be the most structurally compact molecules. All BsAbs were shown to bind both of their target proteins (and corresponding cells) equally well. However, CD19xCD3 IgG2 did not bind both antigens simultaneously as measured by the absence of cellular clustering of T cells with target cells. This translated to a reduced potency of IgG2 BsAbs in T-cell redirection assays. The activity of IgG2 BsAbs was fully restored in the chimeric subclasses IgG4:2 and IgG1:2. This confirmed the major contribution of the F(ab)₂ region to the BsAb’s functional activity and demonstrated that function of BsAbs can be modulated by engineering molecules combining different Fc and F(ab)₂ domains.

Abbreviations: ADCC: Antibody-dependent cellular cytotoxicity; AlphaScreen^TM: Amplified Luminescent Proximity Homogeneous Assay Screening; ANOVA: Analysis of variance; BiTE: bispecific T-cell engager; BSA: bovine serum albumin; BsAb: bispecific antibody; cFAE: controlled Fab-arm exchange; CDC: complement-dependent cellular cytotoxicity; CIEX: cation-exchange; CIR: chimeric immune receptor; DPBS: Dulbecco’s phosphate-buffered saline; EC₅₀ value: effective concentration to reach half-maximum effect; EGFR: epidermal growth factor receptor; EI: expansion index (RA_t=x/RA_t=0); FACS: fluorescence-activated cell sorting; FVD: fixable viability dye; HI-HPLC: hydrophobic interaction HPLC; HI-FBS: heat-inactivated fetal bovine serum; HPLC: high-pressure liquid chromatography; IC₅₀ value: effective concentration to reach half-maximum inhibition; IQ: Inhibition Quotient; IS: immunological synapse; MES: 2-(N-morpholino)ethanesulfonic acid; R-PE: recombinant phycoerythrin; RA: red area in μm²/well; RD: receptor density; RFP: red fluorescent protein; R_g: radius of gyration; RSV: respiratory syncytial virus; SAXS: small-angle x-ray scattering; scFv: single-chain variable fragment; SD: standard deviation; SPR: surface plasmon resonance; WT: wild-type     

Impact of linker and conjugation chemistry on antigen binding,Fc receptor binding and thermal stability of model antibody-drug conjugates

Antibody-drug conjugates (ADCs) with biotin as a model cargo tethered to IgG1 mAbs via different linkers and conjugation methods were prepared and tested for thermostability and ability to bind target antigen and Fc receptor. Most conjugates demonstrated decreased thermostability relative to unconjugated antibody, based on DSC, with carbohydrate and amine coupled ADCs showing the least effect compared with thiol coupled conjugates. A strong correlation between biotin-load and loss of stability is observed with thiol conjugation to one IgG scaffold, but the stability of a second IgG scaffold is relatively insensitive to biotin load. The same correlation for amine coupling was less significant. Binding of antibody to antigen and Fc receptor was investigated using surface plasmon resonance. None of the conjugates exhibited altered antigen affinity. Fc receptor FcγIIb (CD32b) interactions were investigated using captured antibody conjugate. Protein G and Protein A, known inhibitors of Fc receptor (FcR) binding to IgG, were also used to extend the analysis of the impact of conjugation on Fc receptor binding. H10_NPEG4 was the only conjugate to show significant negative impact to FcR binding, which is likely due to higher biotin-load compared with the other ADCs. The ADC aHIS_NLC and aHIS_TPEG8 demonstrated some loss in affinity for FcR, but to much lower extent. The general insensitivity of target binding and effector function of the IgG1 platform to conjugation highlight their utility. The observed changes in thermostability require consideration for the choice of conjugation chemistry, depending on the system being pursued and particular application of the conjugate.     

Modulation of natural cytotoxicity by alloantibodies. V. The mechanism of a selective augmenting effect of anti-H-2 antisera on the natural killer activity of mouse spleen cells

Treatment of mouse spleen cells with specific anti-H-2 antisera augments their natural killer (NK) activity against K562 cells but not against YAC target tumor cells. The same population of natural killer cells was found to lyse K562 as well as YAC target cells, since (a) depletion of YAC reactive NK cells by absorption on YAC monolayers resulted in a concomitant depletion of anti-K562 NK activity of mouse spleen cells, and (b) both K562 and YAC cells could inhibit their own as well as each others lysis in a cross-competition assay. Anti-H-2 antiserum could not induce anti-K562 NK activity in spleen cells previously depleted of NK cells by absorption on YAC monolayers, indicating that alloantiserum does not act by recruiting otherwise nonreactive cells to become cytotoxic toward K562 target cells. In a target-binding assay, K562 binding of NK cells (T-cell-, B-cell-, and macrophage-depleted spleen cells) increased five- to eightfold in the presence of anti-H-2 antiserum whereas YAC cells binding of NK cells was not increased. H-2 antigens per se did not appear to be involved in the alloantisera effect since anti-NK antiserum directed against a non-H-2 antigen selectively expressed on NK cells, showed a similar selective NK enhancing effect. Protein A, a reagent which binds to the Fc region of immunoglobulin molecules, completely blocked the alloantiserum induced augmentation of anti-K562 NK activity, but did not alter basal NK activity. Moreover, the F(ab)₂ fraction of alloantibodies failed to enhance anti-K562 cytotoxic activity of mouse spleen cells, indicating a crucial role for the Fc portion of the alloantibodies attached to the NK cells, in NK augmentation. Utilization of several target cell lines with or without membrane Fc receptors (FcR) revealed that alloantiserum enhanced the lysis of only FcR⁺ target cells. It is proposed that alloantibody-coated NK cells, as a result of a secondary interaction between attached alloantibody and Fc receptors on target cells, interact more readily with the target cells and thereby cause a higher level of lytic activity.     

Overexpression of microRNA-155 increases IL-21 mediated STAT3 signaling and IL-21 production in systemic lupus erythematosus

IntroductionInterleukin (IL)-21 is a key cytokine in autoimmune diseases such as systemic lupus erythematosus (SLE) by its regulation of autoantibody production and inflammatory responses. The objective of this study is to investigate the signaling capacity of IL-21 in T and B cells and assess its possible regulation by microRNA (miR)-155 and its target gene suppressor of cytokine signaling 1 (SOCS1) in SLE.MethodsThe signaling capacity of IL-21 was quantified by stimulating peripheral blood mononuclear cells (PBMCs) with IL-21 and measuring phosphorylation of STAT3 (pSTAT3) in CD4+ T cells, B cells, and natural killer cells. Induction of miR-155 by IL-21 was investigated by stimulating purified CD4+ T cells with IL-21 and measuring miR-155 expression levels. The functional role of miR-155 was assessed by overexpressing miR-155 in PBMCs from SLE patients and healthy controls (HCs) and measuring its effects on STAT3 and IL-21 production in CD4+ and CD8+ T cells.ResultsInduction of pSTAT3 in CD4+ T cells in response to IL-21 was significantly decreased in SLE patients compared to HCs (p < 0.0001). Further, expression levels of miR-155 were significantly decreased and SOCS1 correspondingly increased in CD4+ T cells from SLE patients. Finally, overexpression of miR-155 in CD4+ T cells increased STAT3 phosphorylation in response to IL-21 treatment (p < 0.01) and differentially increased IL-21 production in SLE patients compared to HCs (p < 0.01).ConclusionWe demonstrate that SLE patients have reduced IL-21 signaling capacity, decreased miR-155 levels, and increased SOCS1 levels compared to HCs. The reduced IL-21 signaling in SLE could be rescued by overexpression of miR-155, suggesting an important role for miR-155 in the reduced IL-21 signaling observed in SLE.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0660-z) contains supplementary material, which is available to authorized users.     

Antibody Fc engineering for enhanced neonatal Fc receptor binding and prolonged circulation half-life

ABSTRACT

The neonatal Fc receptor (FcRn) promotes antibody recycling through rescue from normal lysosomal degradation. The binding interaction is pH-dependent with high affinity at low pH, but not under physiological pH conditions. Here, we combined rational design and saturation mutagenesis to generate novel antibody variants with prolonged half-life and acceptable development profiles. First, a panel of saturation point mutations was created at 11 key FcRn-interacting sites on the Fc region of an antibody. Multiple variants with slower FcRn dissociation kinetics than the wildtype (WT) antibody at pH 6.0 were successfully identified. The mutations were further combined and characterized for pH-dependent FcRn binding properties, thermal stability and the FcγRIIIa and rheumatoid factor binding. The most promising variants, YD (M252Y/T256D), DQ (T256D/T307Q) and DW (T256D/T307W), exhibited significantly improved binding to FcRn at pH 6.0 and retained similar binding properties as WT at pH 7.4. The pharmacokinetics in human FcRn transgenic mice and cynomolgus monkeys demonstrated that these properties translated to significantly prolonged plasma elimination half-life compared to the WT control. The novel variants exhibited thermal stability and binding to FcγRIIIa in the range comparable to clinically validated YTE and LS variants, and showed no enhanced binding to rheumatoid factor compared to the WT control. These engineered Fc mutants are promising new variants that are widely applicable to therapeutic antibodies, to extend their circulation half-life with obvious benefits of increased efficacy, and reduced dose and administration frequency.     

Hemagglutination Activity and Localization of Fc Receptor of Group A and G Streptococci

The IgG-Fc binding activity and binding sites on the cell surface of streptococci, strains AR1 (group A) and G148 (group G), and Staphylococcus aureus strain Cowan I were examined by hemagglutination (HA) and immunoelectron microscopic methods. No distinct difference was observed in the HA activity among these three strains. However, the strains differed in the distribution of Fc receptor. Cowan I cells (having protein A) were heavily covered with two layers of ferritin particles, whereas AR1 cells were heavily covered with a single, rough layer of ferritin particles. G148 cells (having protein G) were labeled with a relatively thin, rough ferritin layer. The trypsin susceptibility of the Fc receptors of the AR1 strain was much higher than that of the G148 strain. These results suggest that both streptococcal strains are distinctly different in the arrangement or in the conformation of the Fc receptor from the Cowan I strain. It is also suggested that the Fc receptor molecules of the streptococcal strains differ from each other not only in conformation but also in trypsin susceptibility.     

Signal transduction mechanisms through Fc gamma receptors on the mouse macrophage surface.

Mouse macrophages and macrophage cell lines such as P388D1 or J774 carry at least two distinct Fc gamma receptors (Fc gamma R): one specific for the Fc portion of IgG2a (Fc gamma aR, also classified as Fc gamma RI) and another for IgG2b (Fc gamma 2bR, also classified as Fc gamma RII beta). These Fc gamma Rs should transmit, upon binding of an appropriate ligand, a specific signal that leads to the regulation of macrophage functions, as the interaction of immune complex with cell surface receptor has been shown to lead to suppression of the humoral immune response or B cell differentiation, to the destruction of target cells by antibody-dependent cell-mediated cytotoxicity, to activation of arachidonic acid metabolic cascade, to the phagocytosis of opsonized particles, or to the generation of superoxide anion. In this review, we first describe evidence that Fc gamma 2aR and Fc gamma 2bR are associated with casein kinase II and phospholipase A2 activity, respectively. We will then discuss a potential role for these enzymatic activities in signal transduction pathways that leads to the activation of the arachidonic acid metabolic cascade and adenylate cyclase, to the regulation of phagocytosis, and to the suppression of interferon-gamma action to induce Ia antigens.     

Analysis of Fc(epsilon)RI-mediated mast cell stimulation by surface-carried antigens

Clustering of the type I receptor for IgE (Fc[epsilon]RI) on mast cells initiates a cascade of biochemical processes that result in secretion of inflammatory mediators. To determine the Fc(epsilon)RI proximity, cluster size, and mobility requirements for initiating the Fc(epsilon)RI cascade, a novel experimental protocol has been developed in which mast cells are reacted with glass surfaces carrying different densities of both antigen and bound IgE, and the cell's secretory response to these stimuli is measured. The results have been analyzed in terms of a model based on the following assumptions: 1) the glass surface antigen distribution and consequently that of the bound IgE are random; 2) Fc(epsilon)RI binding to these surface-bound IgEs immobilizes the former and saturates the latter; 3) the cell surface is formally divided into small elements, which function as a secretory stimulus unit when occupied by two or more immobilized IgE-Fc(epsilon)RI complexes; 4) alternatively, similar stimulatory units can be formed by binding of surface-carried IgE dimers to two Fc(epsilon)RI. This model yielded a satisfactory and self-consistent fitting of all of the different experimental data sets. Hence the present results establish the essential role of Fc(epsilon)RI immobilization for initiating its signaling cascade. Moreover, it provides independent support for the notion that as few as two Fc(epsilon)RIs immobilized at van der Waals contact constitute an "elementary stimulatory unit" leading to mast cell (RBL-2H3 line) secretory response.     

The two binding-site models of human IgG binding Fc gamma receptors

Fc receptors (FcR) are immunoglobulin-binding molecules that enable antibodies to perform several biological functions by forming a link between specific antigen recognition and effector cells. FcRs are involved in regulating antibody production as well. Most FcRs belong to the immunoglobulin superfamily, and show structural homology with each other and with their ligands. Recent data on the structure of IgG binding FcRs obtained from monoclonal antibodies and gene cloning studies, as well as on ligand binding capacity and fine specificity of the receptor binding site (or sites), are reviewed. The binding capacity and fine specificity of receptor binding sites, as well as the structure and conformation of the immunoglobulin ligands, play important roles in triggering FcR-mediated signals. In induction of signals, the interaction of the FcR with the CH2 domain of the IgGFc is decisive. The high-affinity Fc gamma RI possess one active binding site specific for contact residues that is located at the N-proximal end of the CH2 domain and is able to mediate both binding and signal transfer. The low-affinity Fc gamma RIII has two active binding sites: the CH3 domain-specific site, which mediates only binding; and the CH2 domain-specific site, which is responsible for binding and signaling. Similarly, the low-affinity Fc gamma RII on resting B cells has one site for CH2 and another for CH3 binding. The expression, release, and fine specificity of Fc gamma RII on B cells correlates with the cell cycle.     

A novel bispecific antibody format enables simultaneous bivalent and monovalent co-engagement of distinct target antigens

Bispecific antibodies based on full-length antibody structures are more optimal than fragment-based formats because they benefit from the favorable properties of the Fc region. However, the homodimeric nature of Fc effectively imposes bivalent binding on all current full-length bispecific antibodies, an attribute that can result in nonspecific activation of cross-linked receptors. We engineered a novel bispecific format, referred to as mAb-Fv, that utilizes a heterodimeric Fc region to enable monovalent co-engagement of a second target antigen in a full-length context. mAb-Fv constructs co-targeting CD16 and CD3 were expressed and purified as heterodimeric species, bound selectively to their co-target antigens and mediated potent cytotoxic activity by NK cells and T cells, respectively. The capacity to co-engage distinct target antigens simultaneously with different valencies is an improved feature for bispecific antibodies with promising therapeutic implications.Key words: bispecific, mAb-Fv, Fc, heterodimer, CD16, CD3, HER2, HM1.24, anti-tumor, cancerDespite the enormous success of antibody-based therapeutics for the treatment of a variety of diseases, research efforts to improve their clinical efficacy continue. One avenue being explored is the engineering of new antigen binding sites to permit co-engagement of two distinct targets. Such engineered antibodies are commonly referred to as bispecifics, and a wide variety of formats have been described in references ¹ and ². Co-target antigens can include two targets believed to be causal in the pathology of a particular disease, e.g., two cytokines or growth factors.^3–5 Alternatively, the co-target pair may be a cell surface antigen and an immune receptor such that a novel “effector” mechanism can be built into the antibody, beyond those mediated naturally by the Fc region.²In the 1980s, bispecific antibodies were made by fusing two cell lines that each produced a single monoclonal antibody (mAb).⁶ Although the resulting hybrid hybridoma or quadroma did produce bispecifics, they were only a minor population and extensive purification was required to isolate the desired antibody. Antibody fragments provided an engineering solution to this problem; because they lack the complex quaternary structure of a full-length antibody, multiple variable regions can be linked in single genetic constructs. Antibody fragments of many different forms have been generated, including diabodies, single chain diabodies, tandem scFvs and F(ab'')₂ bispecifics.^2,7 While these formats can be expressed at high levels in bacteria and, arguably, may have benefits due to their small size, they suffer from poor half-life in vivo and can present manufacturing challenges related to their production and stability. For example, the rapid clearance of some fragment-based bispecifics requires that they be infused continuously via a portable pump over one to two months.⁸ The principal source of these limitations for fragment formats is the lack of an antibody Fc region with its associated structural and functional benefits, including large size that precludes renal filtration; high stability; binding to various Fc ligands, one of which maintains serum persistence (the neonatal Fc receptor FcRn) and binding to proteins A and G, which facilitates large scale purification.Recent work has attempted to address the shortcomings of fragment-based bispecifics by engineering a second antigen binding site into full-length antibody-like formats.^5,9–12 The presence of an Fc region in theory provides these formats with the developability and pharmacokinetic properties of standard IgG mAbs. However, because these constructs build new antigen binding sites on top of a homodimeric constant chain, binding to the new antigen is always bivalent. This consequence may pose a constraint depending on the co-targeting goal.For many immune receptors, cellular activation is accomplished by cross-linking of a monovalent binding interaction. The mechanism of cross-linking is typically mediated by antibody/antigen immune complexes, or via effector cell to target cell engagement. For example, the low affinity activating Fc gamma receptors (FcγRs) such as CD16 (FcγRIIIa) and CD32a (FcγRIIa) that mediate cellular killing bind monovalently to the antibody Fc region. While monovalent binding does not result in cellular signaling, upon effector cell engagement with the target cell, receptors are cross-linked and clustered on the cell surface, leading to activation.¹³ On T cells, CD3 activation occurs when its associated T-cell receptor (TCR) engages antigen-loaded major histocompatibility complex (MHC) on antigen-presenting cells in an avid cell-to-cell synapse.¹⁴ Bivalent antibodies targeting CD3 can elicit massive cytokine release, and the consequent toxicity has presented challenges for the development of anti-CD3 antibodies as drugs;^15,16 in contrast, monovalent binding of CD3 in Fab^17,18 and bispecific¹⁹ formats generates much lower levels of T-cell activation. For bispecifics, a consequence of this biology is that bivalent cross-linking of receptors can lead to non-specific activation of an effector cell in the absence of target cell.Thus, when the therapeutic goal is the co-engagement of an immune receptor, the desired binding may be monovalent rather than bivalent. This mode is incompatible with the majority of current full-length bispecifics. We describe an engineering solution to this problem that utilizes a heterodimeric Fc region to enable a single additional variable region to be built monomerically onto an antibody. Our new bispecific format, which we refer to as mAb-Fv, enables the simultaneous bivalent and monovalent co-engagement of distinct target antigens.