Insights from Fc receptor biology: A route to improved antibody reagents

Fc receptors and their interaction with antibodies will be a major theme at the forthcoming FASEB Science Research Conference on Immunoreceptors to be held in Snowmass this July (details available at www.faseb.org/src/home.aspx, follow the tabs for Immunoreceptors). Since its inception in the mid 1980s, this meeting series has maintained a focus on Fc receptors, and this year’s meeting will be no exception.     

Crystal structure of the HSV-1 Fc receptor bound to Fc reveals a mechanism for antibody bipolar bridging

Herpes simplex virus type-1 expresses a heterodimeric Fc receptor, gE-gI, on the surfaces of virions and infected cells that binds the Fc region of host immunoglobulin G and is implicated in the cell-to-cell spread of virus. gE-gI binds immunoglobulin G at the basic pH of the cell surface and releases it at the acidic pH of lysosomes, consistent with a role in facilitating the degradation of antiviral antibodies. Here we identify the C-terminal domain of the gE ectodomain (CgE) as the minimal Fc-binding domain and present a 1.78-Å CgE structure. A 5-Å gE-gI/Fc crystal structure, which was independently verified by a theoretical prediction method, reveals that CgE binds Fc at the C _H2-C _H3 interface, the binding site for several mammalian and bacterial Fc-binding proteins. The structure identifies interface histidines that may confer pH-dependent binding and regions of CgE implicated in cell-to-cell spread of virus. The ternary organization of the gE-gI/Fc complex is compatible with antibody bipolar bridging, which can interfere with the antiviral immune response.     

GABAA receptor subtypes: from pharmacology to molecular biology.

GABAA receptors are GABA (gamma-aminobutyric acid)-gated chloride channels, which are major mediators of neuronal inhibition in the brain and are modulated by benzodiazepines, barbiturates, alcohol, and other important centrally acting drugs. Although previous pharmacological and biochemical data had suggested a degree of heterogeneity, recent cloning of at least 15 different receptor subunits, thought to be combined in groups of five, indicates that the brain may contain a truly astonishing variety of GABAA receptor subtypes. This review describes the little that is known about these subtypes, emphasizing possible molecular bases of receptor heterogeneity. We also discuss approaches to establishing the subunit composition of subtypes.     

Impact of SPR biosensor assay configuration on antibody: Neonatal Fc receptor binding data

Binding interactions with the neonatal Fc receptor (FcRn) are one determinant of pharmacokinetic properties of recombinant human monoclonal antibody (rhumAb) therapeutics, and a conserved binding motif in the crystallizable fragment (Fc) region of IgG molecules interacts with FcRn. Surface plasmon resonance (SPR) biosensor assays are often used to characterize interactions between FcRn and rhumAb therapeutics. In such assays, generally either the rhumAb (format 1) or the FcRn protein (format 2) is immobilized on a biosensor chip. However, because evidence suggests that, in some cases, the variable domains of a rhumAb may also affect FcRn binding, we evaluated the effect of SPR assay configuration on binding data. We sought to assess FcRn binding properties of 2 rhumAbs (rhumAb1 and rhumAb2) to FcRn proteins using these 2 biosensor assay formats. The two rhumAbs have greater than 99% sequence identity in the Fc domain but differ in their Fab regions. rhumAb2 contains a positively charged patch in the variable domain that is absent in rhumAb1. Our results showed that binding of rhumAb1 to FcRn was independent of biosensor assay configuration, while binding of rhumAb2 to FcRn was highly SPR assay configuration dependent. Further investigations revealed that the format dependency of rhumAb2-FcRn binding is linked to the basic residues that form a positively charged patch in the variable domain of rhumAb2. Our work highlights the importance of analyzing rhumAb-FcRn binding interactions using 2 alternate SPR biosensor assay configurations. This approach may also provide a simple way to identify the potential for non-Fc-driven FcRn binding interactions in otherwise typical IgGs.     

Shortened engineered human antibody CH2 domains: increased stability and binding to the human neonatal Fc receptor

The immunoglobulin (Ig) constant CH2 domain is critical for antibody effector functions. Isolated CH2 domains are promising scaffolds for construction of libraries containing diverse binders that could also confer some effector functions. We have shown previously that an isolated human CH2 domain is relatively unstable to thermally induced unfolding, but its stability can be improved by engineering an additional disulfide bond (Gong, R., Vu, B. K., Feng, Y., Prieto, D. A., Dyba, M. A., Walsh, J. D., Prabakaran, P., Veenstra, T. D., Tarasov, S. G., Ishima, R., and Dimitrov, D. S. (2009) J. Biol. Chem. 284, 14203-14210). We have hypothesized that the stability of this engineered antibody domain could be further increased by removing unstructured residues. To test our hypothesis, we removed the seven N-terminal residues that are in a random coil as suggested by our analysis of the isolated CH2 crystal structure and NMR data. The resulting shortened engineered CH2 (m01s) was highly soluble, monomeric, and remarkably stable, with a melting temperature (T(m)) of 82.6 °C, which is about 10 and 30 °C higher than those of the original stabilized CH2 (m01) and CH2, respectively. m01s and m01 were more resistant to protease digestion than CH2. A newly identified anti-CH2 antibody that recognizes a conformational epitope bound to m01s significantly better (>10-fold higher affinity) than to CH2 and slightly better than to m01. m01s bound to a recombinant soluble human neonatal Fc receptor at pH 6.0 more strongly than CH2. These data suggest that shortening the m01 N terminus significantly increases stability without disrupting its conformation and that our approach for increasing stability and decreasing size by removing unstructured regions may also apply to other proteins.     

Functional optimization of agonistic antibodies to OX40 receptor with novel Fc mutations to promote antibody multimerization

Immunostimulatory receptors belonging to the tumor necrosis factor receptor (TNFR) superfamily are emerging as promising targets for cancer immunotherapies. To optimize the agonism of therapeutic antibodies to these receptors, Fc engineering of antibodies was applied to facilitate the clustering of cell surface TNFRs to activate downstream signaling pathways. One engineering strategy is to identify Fc mutations that facilitate antibody multimerization on the cell surface directly. From the analyses of the crystal packing of IgG1 structures, we identified a novel set of Fc mutations, T437R and K248E, that facilitated antibody multimerization upon binding to antigens on cell surface. In a NF-κB reporter assay, the engineered T437R/K248E mutations could facilitate enhanced agonism of an anti-OX40 antibody without the dependence on FcγRIIB crosslinking. Nonetheless, the presence of cells expressing FcγRIIB could facilitate a boost of the agonism of the engineered antibody with mutations on IgG1 Fc, but not on the silent IgG2σ Fc. The Fc engineered antibody also showed enhanced effector functions, including antibody-dependent cell-meditated cytotoxicity, antibody-dependent cellular phagocytosis, and complement-dependent cytotoxicity, depending on the IgG subtypes. Also, the engineered antibodies showed normal FcRn binding and pharmacokinetic profiles in mice. In summary, this study elucidated a novel Fc engineering approach to promote antibody multimerization on a cell surface, which could enhance agonism and improve effector function for anti-TNFR antibodies as well as other therapeutic antibodies.     

Insights into neural stem cell biology from flies

Drosophila neuroblasts are similar to mammalian neural stem cells in their ability to self-renew and to produce many different types of neurons and glial cells. In the past two decades, great advances have been made in understanding the molecular mechanisms underlying embryonic neuroblast formation, the establishment of cell polarity and the temporal regulation of cell fate. It is now a challenge to connect, at the molecular level, the different cell biological events underlying the transition from neural stem cell maintenance to differentiation. Progress has also been made in understanding the later stages of development, when neuroblasts become mitotically inactive, or quiescent, and are then reactivated postembryonically to generate the neurons that make up the adult nervous system. The ability to manipulate the steps leading from quiescence to proliferation and from proliferation to differentiation will have a major impact on the treatment of neurological injury and neurodegenerative disease.     

Insights from comprehensive multiple receptor docking to HDAC8

A systematic investigation of the available crystal structures of HDAC8 and of the influence of different receptor structures and docking protocols is presented. The study shows that the open conformation of HDAC8 may be preferred by ligands with flexible surface binding groups, as such a conformation allows the ligands to minimize their exposure to solvent upon binding. This observation allowed us to rationalize the excellent potency of pyrazole-based inhibitors compared to that of isoxazole-based inhibitors.     

Fc receptor dependency of antibody mediated feedback regulation: on the mechanism of inhibition.

Culture conditions were developed to obtain long-lasting immune responses in vitro with horse lymph nodes. Immunologic memory can be elicited by primary antigenic stimulation in test tubes at the outset of the cultures and can be detected 3 weeks thereafter. Generation of memory can occur in the absence of detectable antibody synthesis. The data, which agree with previous observations in vivo, indicate that memory and antibodies can be produced independently and that generation of memory is the first of these events to occur after a priming antigenic contact.     

IgG Fc engineering to modulate antibody effector functions

Therapeutic monoclonal antibodies are among the most effective biotherapeutics to date. An important aspect of antibodies is their ability to bind antigen while at the same time recruit immune effector functions. The majority of approved recombinant monoclonal antibody therapies are of the human IgG1 subclass, which can engage both humoral and cellular components of the immune system. The wealth of information generated about antibodies has afforded investigators the ability to molecularly engineer antibodies to modulate effector functions. Here, we review various antibody engineering efforts intended to improve efficacy and safety relative to the human IgG isotype. Further, we will discuss proposed mechanisms by which engineering approaches led to modified interactions with immune components and provide examples of clinical studies using next generation antibodies.     

The murine lymphocyte receptor for IgE. III. Use of chemical cross-linking reagents to further characterize the B lymphocyte Fc epsilon receptor

Cross-linking reagents were used to further characterize the murine B cell receptor for the Fc portion of IgE (Fc epsilon R) and compare this receptor to the well-characterized high-affinity Fc epsilon R on rat basophilic leukemia (RBL) cells. The disulfide cleavable and noncleavable reagents 3,3'-dithiobis(sulfosuccinimidyl) propionate (DTSSP) and bis(sulfosuccinimidyl) suberate (BS3) were used. With these reagents, efficient cross-linking of the alpha component of the high-affinity RBL Fc epsilon R to the membrane-buried beta and gamma components occurred only if the membrane was solubilized before the cross-linking reaction. In studies with purified murine B cells, IgE could be cross-linked to the Fc epsilon R on intact cells with either DTSSP or BS3. Under the same conditions, up to 10% of the B cell surface immunoglobulin (sIg) (both IgM and IgD) was also found to cross-link to a portion of the IgE/Fc epsilon R complex, suggesting that on the intact murine B cell the Fc epsilon R is frequently in close association with sIg. The B cell Fc epsilon R was also examined for the presence of receptor-associated proteins. Under conditions where the high-affinity RBL Fc epsilon R was substantially cross-linked to the alpha, beta, gamma complex, no evidence was seen for similar cross-linking of the B cell Fc epsilon R. Cross-linking experiments on affinity-purified Fc epsilon R preparations also gave no evidence for receptor-associated proteins with the B cell Fc epsilon R, although evidence for receptor-receptor association was seen. Thus, these data further support the concept that there may be little relationship between the high-affinity mast cell/basophil Fc epsilon R and the low-affinity lymphocyte Fc epsilon R.     

Novel antibody reagents: Production and potential

Immunologists have had limited control over the compositions of monoclonal antibodies. They could choose the cell lines to be fused and screen hybridomas for the production of antibodies with an appropriate specificity. Recently, however, the degree of control has been extended by advances in cell fusion and genetic engineering. In particular, monoclonal antibodies with dual specificities, predetermined specificities or additional functional moieties can be produced.     

A monoclonal antibody to human insulin receptor

A murine hybridoma secreting antibody against human insulin receptor was produced by fusing FO myeloma cells with spleen and lymph node cells from a mouse that had been immunized with insulin receptor purified from human placenta. The secreted antibody was an IgG₁ (κ), designated αIR-1. Like the previously described rabbit polyclonal antibody, αIR-1 did not inhibit insulin binding. It specifically immunoprecipitated ¹²⁵I-insulin-receptor complexes as well as unoccupied receptor previously labeled directly with lactoperoxidase. Thus, αIR-1 interacts with the receptor at a site distinct from the insulin binding site. Unlike previously described anti-insulin receptor antibodies, αIR-1 exhibits strong tissue and species specificity.     

The IgG Fc contains distinct Fc receptor (FcR) binding sites: the leukocyte receptors Fc gamma RI and Fc gamma RIIa bind to a region in the Fc distinct from that recognized by neonatal FcR and protein A

The CH2-CH3 interface of the IgG Fc domain contains the binding sites for a number of Fc receptors including Staphylococcal protein A and the neonatal Fc receptor (FcRn). It has recently been proposed that the CH2-CH3 interface also contains the principal binding site for an isoform of the low affinity IgG Fc receptor II (Fc gamma RIIb). The Fc gamma RI and Fc gamma RII binding sites have previously been mapped to the lower hinge and the adjacent surface of the CH2 domain although contributions of the CH2-CH3 interface to binding have been suggested. This study addresses the question whether the CH2-CH3 interface plays a role in the interaction of IgG with Fc gamma RI and Fc gamma RIIa. We demonstrate that recombinant soluble murine Fc gamma RI and human Fc gamma RIIa did not compete with protein A and FcRn for binding to IgG, and that the CH2-CH3 interface therefore appears not to be involved in Fc gamma RI and Fc gamma RIIa binding. The importance of the lower hinge was confirmed by introducing mutations in the proposed binding site (LL234,235AA) which abrogated binding of recombinant soluble Fc gamma RIIa to human IgG1. We conclude that the lower hinge and the adjacent region of the CH2 domain of IgG Fc is critical for the interaction between Fc gamma RIIa and human IgG, whereas contributions of the CH2-CH3 interface appear to be insignificant.     

Leishmania donovani: generation of monospecific antibody reagents to soluble acid phosphatase

Four monoclonal antibodies (McAbs) were generated against the soluble extracellular acid phosphatase (EC 3.1.3.2) (S-AcP) of Leishmania donovani. These were detected in the primary screen using an ELISA with promastigote culture supernatants as antigen. Three of the McAbs demonstrated bound S-AcP from such culture supernatants in an enzyme activity binding assay. All immunoprecipitated metabolically labeled S-AcP but none showed any binding to the promastigote surface by indirect immunofluorescence. Moreover, none reacted with Triton X-100 solubilized plasma membranes by immunoprecipitation or Western blotting. These results demonstrated that the McAbs did not recognize the surface membrane bound acid phosphatase, but were specific for the extracellular soluble enzyme. Further, none of the antibodies immunoprecipitated any of the five human acid phosphatase isozymes or reacted with them in Western blots or the enzyme activity binding assay. Therefore, they are specific for the parasite-derived enzyme. One of these was used to affinity purify sufficient L. donovani S-AcP to immunize a rabbit and generate a specific, polyvalent antiserum. This polyvalent antibody immunoprecipitated S-AcP activity but did not cross-react with the surface membrane acid phosphatase, indicating that these two parasite enzymes are separate gene products.     

Charge heterogeneity: Basic antibody charge variants with increased binding to Fc receptors

We identified active isoforms of the chimeric anti-GD2 antibody, ch14.18, a recombinant antibody produced in Chinese hamster ovary cells, which is already used in clinical trials.^1,2,3 Ladenstein R, Weixler S, Baykan B, Bleeke M, Kunert R, Katinger D, Pribill I, Glander P, Bauer S, Pistoia V, et al. Ch14.18 antibody produced in CHO cells in relapsed or refractory Stage 4 neuroblastoma patients: a SIOPEN Phase 1 study. MAbs 2013; 5:801-9; PMID:23924804; http://dx.doi.org/10.4161/mabs.25215 Desai AV, Fox E, Smith LM, Lim AP, Maris JM, Balis FM. Pharmacokinetics of the chimeric anti-GD2 antibody, ch14.18, in children with high-risk neuroblastoma. Cancer Chemother Pharmacol 2014; 74:1047-55; PMID:25212536; http://dx.doi.org/10.1007/s00280-014-2575-9 Siebert N, Eger C, Seidel D, Jüttner M, Zumpe M, Wegner D, Kietz S, Ehlert K, Veal GJ, Siegmund W, et al. Pharmacokinetics and pharmacodynamics of ch14.18/CHO in relapsed/refractory high-risk neuroblastoma patients treated by long-term infusion in combination with IL-2. MAbs 2016; 8:604-16; PMID:26785755; http://dx.doi.org/10.1080/19420862.2015.1130196  We separated the antibody by high resolution ion-exchange chromatography with linear pH gradient elution into acidic, main and basic charge variants on a preparative scale yielding enough material for an in-depth study of the sources and the effects of microheterogeneity. The binding affinity of the charge variants toward the antigen and various cell surface receptors was studied by Biacore. Effector functions were evaluated using cellular assays for antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. Basic charge variants showed increased binding to cell surface receptor FcγRIIIa, which plays a major role in regulating effector functions. Furthermore, increased binding of the basic fractions to the neonatal receptor was observed. As this receptor mediates the prolonged half-life of IgG in human serum, this data may well hint at an increased serum half-life of these basic variants compared to their more acidic counterparts. Different glycoform patterns, C-terminal lysine clipping and N-terminal pyroglutamate formation were identified as the main structural sources for the observed isoform pattern. Potential differences in structural stability between individual charge variant fractions by nano differential scanning calorimetry could not been detected. Our in-vitro data suggests that the connection between microheterogeneity and the biological activity of recombinant antibody therapeutics deserves more attention than commonly accepted.     

IgG-mediated enhancement of antibody responses is low in Fc receptor gamma chain-deficient mice and increased in Fc gamma RII-deficient mice.

Immunization with IgG/Ag or IgE/Ag complexes leads to a higher production of specific Abs than immunization with Ag alone. The enhancing effect of IgE is exclusively dependent upon the low-affinity receptor for IgE, Fc epsilon RII, whereas the mechanism behind IgG-mediated enhancement is unknown. We have investigated whether receptors for the Fc part of IgG are required for responses to IgG/Ag. Mice lacking the gamma subunit of Fc receptors (FcRs) (FcR gamma-/-), Fc gamma RII (Fc gamma RII-/-), or Fc gamma RIII (Fc gamma RIII-/-) were immunized with BSA-2,4,6-trinitrophenyl (TNP) alone or BSA-TNP complexed to monoclonal TNP-specific IgG1, IgG2a, or IgG2b. As expected, all subclasses enhanced the Ab-response to BSA in wild-type mice. Enhancement was in the same order of magnitude in Fc gamma RIII-/- mice (相似文献