Michael L. Doyle

HIV-1 evades antibody-mediated neutralization through conformational masking of receptor-binding sites

The ability of human immunodeficiency virus (HIV-1) to persist and cause AIDS is dependent on its avoidance of antibody-mediated neutralization. The virus elicits abundant, envelope-directed antibodies that have little neutralization capacity. This lack of neutralization is paradoxical, given the functional conservation and exposure of receptor-binding sites on the gp120 envelope glycoprotein, which are larger than the typical antibody footprint and should therefore be accessible for antibody binding. Because gp120–receptor interactions involve conformational reorganization, we measured the entropies of binding for 20 gp120-reactive antibodies. Here we show that recognition by receptor-binding-site antibodies induces conformational change. Correlation with neutralization potency and analysis of receptor–antibody thermodynamic cycles suggested a receptor-binding-site ‘conformational masking’ mechanism of neutralization escape. To understand how such an escape mechanism would be compatible with virus–receptor interactions, we tested a soluble dodecameric receptor molecule and found that it neutralized primary HIV-1 isolates with great potency, showing that simultaneous binding of viral envelope glycoproteins by multiple receptors creates sufficient avidity to compensate for such masking. Because this solution is available for cell-surface receptors but not for most antibodies, conformational masking enables HIV-1 to maintain receptor binding and simultaneously to resist neutralization.

Elimination of Fc receptor-dependent effector functions of a modified IgG4 monoclonal antibody to human CD4.

Several CD4 mAbs have entered the clinic for the treatment of autoimmune diseases or transplant rejection. Most of these mAbs caused CD4 cell depletion, and some were murine mAbs which were further hampered by human anti-mouse Ab responses. To obviate these concerns, a primatized CD4 mAb, clenoliximab, was generated by fusing the V domains of a cynomolgus macaque mAb to human constant regions. The heavy chain constant region is a modified IgG4 containing two single residue substitutions designed to ablate residual Fc receptor binding activity and to stabilize heavy chain dimer formation. This study compares and contrasts the in vitro properties of clenoliximab with its matched IgG1 derivative, keliximab, which shares the same variable regions. Both mAbs show potent inhibition of in vitro T cell responses, lack of binding to complement component C1q, and inability to mediate complement-dependent cytotoxicity. However, clenoliximab shows markedly reduced binding to Fc receptors and therefore does not mediate Ab-dependent cell-mediated cytotoxicity or modulation/loss of CD4 from the surface of T cells, except in the presence of rheumatoid factor or activated monocytes. Thus, clenoliximab retains the key immunomodulatory attributes of keliximab without the liability of strong Fcγ receptor binding. In initial clinical trials, these properties have translated to a reduced incidence of CD4+ T cell depletion.

Mutagenic Stabilization and/or Disruption of a CD4-Bound State Reveals Distinct Conformations of the Human Immunodeficiency Virus Type 1 gp120 Envelope Glycoprotein

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) gp120 exterior envelope glycoprotein is conformationally flexible. Upon binding to the host cell receptor CD4, gp120 assumes a conformation that is recognized by the second receptor, CCR5 and/or CXCR4, and by the CD4-induced (CD4i) antibodies. Guided by the X-ray crystal structure of a gp120-CD4-CD4i antibody complex, we introduced changes into gp120 that were designed to stabilize or disrupt this conformation. One mutant, 375 S/W, in which the tryptophan indole group is predicted to occupy the Phe 43 cavity in the gp120 interior, apparently favors a gp120 conformation closer to that of the CD4-bound state. The 375 S/W mutant was recognized as well as or better than wild-type gp120 by CD4 and CD4i antibodies, and the large decrease in entropy observed when wild-type gp120 bound CD4 was reduced for the 375 S/W mutant. The recognition of the 375 S/W mutant by CD4BS antibodies, which are directed against the CD4-binding region of gp120, was markedly reduced compared with that of the wild-type gp120. Compared with the wild-type virus, viruses with the 375 S/W envelope glycoproteins were resistant to neutralization by IgG1b12, a CD4BS antibody, were slightly more sensitive to soluble CD4 neutralization and were neutralized more efficiently by the 2G12 antibody. Another mutant, 423 I/P, in which the gp120 bridging sheet was disrupted, did not bind CD4, CCR5, or CD4i antibodies, even though recognition by CD4BS antibodies was efficient. These results indicate that CD4BS antibodies recognize conformations of gp120 different from that recognized by CD4 and CD4i antibodies.

A Direct Comparison of the Activities of Two Humanized Respiratory Syncytial Virus Monoclonal Antibodies: MEDI-493 and RSHZl9

Two humanized monoclonal antibodies, MEDI-493 and RSHZ19, were developed independently as potential improvements over RSV-IGIV for prevention of respiratory syncytial virus (RSV) infection. RSV-IGIV is a polyclonal human antibody preparation for intravenous infusion enriched for RSV neutralizing activity. A phase III clinical trial showed that MEDI-493 significantly reduced hospitalizations due to RSV infection. In a separate trial, RSHZ19 failed to show significant efficacy. In new studies, the in vitro and in vivo activities of MEDI-493 and RSHZ19 were compared to determine whether the different clinical results are related to differences in biologic activity. MEDI-493 was consistently 4- to 5-fold more potent than RSHZ19 in antigen binding, RSV neutralization, and fusion inhibition assays. Although both MEDI-493 and RSHZ19 were effective against A and B subtypes of RSV in the cotton rat model of RSV infection, 2- to 4-fold higher doses of RSHZ19 were required for similar protection. The enhanced activity of MEDI-493 compared with RSHZ19 may, in part, explain its better clinical effect.

Binding Interactions of Human Interleukin 5 with Its Receptor α Subunit LARGE SCALE PRODUCTION, STRUCTURAL, AND FUNCTIONAL STUDIES OF DROSOPHILA-EXPRESSED RECOMBINANT PROTEINS

Human interleukin 5 (hIL5) and soluble forms of its receptor α subunit were expressed in Drosophila cells and purified to homogeneity, allowing a detailed structural and functional analysis. B cell proliferation confirmed that the hIL5 was biologically active. Deglycosylated hIL5 remained active, while similarly deglycosylated receptor α subunit lost activity. The crystal structure of the deglycosylated hIL5 was determined to 2.6-Å resolution and found to be similar to that of the protein produced in Escherichia coli. Human IL5 was shown by analytical ultracentrifugation to form a 1:1 complex with the soluble domain of the hIL5 receptor α subunit (shIL5Rα). Additionally, the relative abundance of ligand and receptor in the hIL5·shIL5Rα complex was determined to be 1:1 by both titration calorimetry and SDS-polyacrylamide gel electrophoresis analysis of dissolved cocrystals of the complex. Titration microcalorimetry yielded equilibrium dissociation constants of 3.1 and 2.0 n M, respectively, for the binding of hIL5 to shIL5Rα and to a chimeric form of the receptor containing shIL5Rα fused to the immunoglobulin Fc domain (shIL5Rα-Fc). Analysis of the binding thermodynamics of IL5 and its soluble receptor indicates that conformational changes are coupled to the binding reaction. Kinetic analysis using surface plasmon resonance yielded data consistent with the Kdvalues from calorimetry and also with the possibility of conformational isomerization in the interaction of hIL5 with the receptor α subunit. Using a radioligand binding assay, the affinity of hIL5 with full-length hIL5Rα in Drosophila membranes was found to be 6 n M, in accord with the affinities measured for the soluble receptor forms. Hence, most of the binding energy of the α receptor is supplied by the soluble domain. Taken with other aspects of hIL5 structure and biological activity, the data obtained allow a prediction for how 1:1 stoichiometry and conformational change can lead to the formation of hIL5·receptor αβ complex and signal transduction.

[8] Tight binding affinities determined from thermodynamic linkage to protons by titration calorimetry

A general titration calorimetry method is described that can be used to determine the affinity of tight binding interactions with proteins. The method is based on the thermodynamic linkage between ligand binding and coupled protonation reactions. The protons linked to a given ligand-binding reaction are measured by titration calorimetry, and integration of the resulting data set yields the pH dependence of the binding affinity based on thermodynamic relationships developed elsewhere. When the pH dependence of the binding affinity is combined with the absolute affinity determined independently at a pH at which the affinity can be conveniently measured, the absolute binding affinity over the entire pH range is determined. The method is well suited for determining high-affinity binding interactions of protein antigens with antibodies, but is applicable to any macromolecular ligand-binding reaction that is coupled to protonation.

Effects of NaCl on the linkages between O2 binding and subunit assembly in human hemoglobin: titration of the quaternary enhancement effect

Oxygen binding by human hemoglobin (Hb) and the coupled reactions of dimer-tetramer assembly were studied over a range of NaCl concentrations (from 0.08 M to 1.4 M) at pH 7.4 and 21.5 degrees C. A strategy of multi-dimensional analysis was employed [G.K. Ackers and H.R. Halvorson, Proc. Natl. Acad. Sci. U.S.A., 91, (1974) 4312] to optimize the resolution of the contributions to cooperativity and their heterotropic salt linkages at each stoichiometric degree of O2 binding. A wide range of Hb concentration was utilized at each [NaCl] in which O2-linked subunit assembly reactions contributed significantly to the positions and shapes of the binding isotherms. Kinetic determinations yielded forward and reverse rate constants for assembly of the unligated species. Amplitudes for the assembly rate data had concentration dependences in agreement with the independently determined dimer-tetramer assembly constants of oxyhemoglobin. Concentration-dependent binding isotherms were analyzed, in combination with the kinetically determined equilibrium constants, to yield salt-linked components of cooperativity at the four stages of oxygenation. The principal results of this study were as follows. (i) Assembly of fully oxygenated Hb tetramers is opposed by NaCl: the dimer-to-tetramer equilibrium constant becomes two orders of magnitude less favorable over the [NaCl] range 0.08 M to 1.4 M. By contrast, for deoxy-Hb the assembly equilibrium constant is reduced only two-fold. (ii) Oxygen binding to dimers is non-cooperative over the entire salt range, whereas dimer affinity is slightly favored by increasing the NaCl concentration. (iii) Overall affinity of tetramers for O2 is opposed by NaCl, becoming an order of magnitude less favorable over the range employed. Most of this decrease occurs at the fourth binding step, which shows a large, salt-mediated quaternary enhancement effect; i.e., the assembly of dimers into tetramers at 0.08 M NaCl is accompanied by an eight-fold increase in O2 affinity. (iv) The quaternary enhancement effect at the last O2-binding step is titrated progressively by salt until it reaches a negligible value near the highest [NaCl] of this study. The lowest [NaCl] condition (0.08 M) elicits the greatest tetramer cooperativity with the largest maximal Hill coefficient and the greatest suppression of intermediates. Possible origins and mechanistic implications of these phenomena are considered.

Measurement of protein interaction bioenergetics: Application to structural variants of anti-sCD4 antibody

This chapter has described a bioenergetic analysis of the interaction of sCD4 with an IgG1 and two IgG4 derivatives of an anti-sCD4 MAb. The MAbs have identical VH and VL domains but differ markedly in their CH and CL domains, raising the question of whether their antigen-binding chemistries are altered. We find the sCD4-binding kinetics and thermodynamics of the MAbs are indistinguishable, which indicates rigorously that the molecular details of the binding interactions are the same. We also showed the importance of using multiple biophysical methods to define the binding model before the bioenergetics can be appropriately interpreted. Analysis of the binding thermodynamics and kinetics suggests conformational changes that might be coupled to sCD4 binding by these MAbs are small or absent.

Evaluating energetics of erythropoietin ligand binding to homodimerized receptor extracellular domains

Abstract A number of techniques have been employed to characterize the energetics of EPO-EPOR-Fc interactions. AUC studies have shown that EPO and EPOR-Fc exist as monomers at concentrations less that 10 μM. Under these conditions, EPO and the EPOR-Fc associate to form a 1:1 complex and this complex does not undergo any further assembly processes. Studies in which the biological activity of EPO at a cell surface is competed by free and dimerized receptor show that the dimerized receptor is 750-fold more potent. This suggests that EPO is bound by both receptor subunits on the Fc chimera, as shown in Fig. 9D. This assembly model provides a foundation for interpretation of the kinetic, thermodynamic, and spectral results. SPR kinetic analyses of the EPO-EPOR-Fc interaction yields association and dissociation rate constants of 8.0 × 10 7 M −1 sec −1 and 2.4 × 10 −4 sec −1 , respectively, for an overall affinity of 3 p M (see Fig. 12). The half-maximal response in a cellular proliferation assay is evoked at an EPO concentration of 10 p M , 54 which is similar to the affinity kinetically determined for the EPOR-Fc. This value suggests that the EPOR-Fc chimera may be a reasonable model for the receptor on a cell surface (see Fig. 17). The use of this reagent is also supported by the studies of Remy et al. , who demonstrate that the EPOR is likely to exist as a dimer on the cell surface, in the absence of ligand. Titration calorimetry confirms the 1:1 stoichiometry, observed by AUC and SPR approaches. Further, the temperature dependence of the enthalpy yields a heat capacity that can be interpreted in terms of a large conformational change in the EPOR on EPO binding. Comparing the structures of the free and complexed receptor, some conformational changes are noted in loops L3 and L6. 18 However, these changes are small compared with the conformational changes predicted from an analysis of the calorimetric data reported here, i.e., equivalent to the folding of ∼70 amino acids. The change in buried surface area between the free and complexed EPOR, determined from structural data, is also quite small when compared with the predicted value of 7500 A 2 from calorimetry. Further studies need to be done to rationalize these observations. These may include an attempt to determine if conformational changes are communicated to the Fc domain and the extent to which EPOR extracellular domains are oriented on the Fc domain in a manner that faithfully reflects their orientation on a cell surface. Finally, while changes in the CD spectra are observed on binding of EPO to the EPOR-Fc, and the monomeric receptor, these changes may be due to subtle changes in the microenvironments of tryptophans and tyrosines and do not require conformational changes of the magnitude suggested from the calorimetry results. In summary, to define macromolecular interactions in solution, the stoichiometry, thermodynamics, and kinetics of assembly need to be understood. This task requires that a multitechnology approach be implemented. Here, AUC established an assembly model and provided a foundation on which SPR, ITC, and CD studies could be based and from which interpretation of these data could be extended. SPR established that the affinity of the dimerized receptor was high and ITC suggested that there may be a significant conformational change on binding. CD suggested that observed spectral changes may be due to these presumed conformational changes, but would also be consistent with more subtle changes. These studies further demonstrate that the EPOR-Fc is a valid model for the dimerized receptor on the cell surface and, as such, will be a useful tool for probing the differences in the interactions of the receptor dimer with EPO agonists and antogonists.

Experimental Dissection of Protein-Protein Interactions in Solution

Publisher Summary Protein–protein interactions play crucial roles in a wide variety of processes, which define cell structure and function. This chapter describes the emerging tools and strategies for determining the kinetics and thermodynamics of protein–protein interactions. These biophysical methods can be used to play dominant roles as core tools for studying the structural and functional molecular properties of protein–protein interactions. In general, these methods require relatively small amounts (milligrams) of protein material and are universally applicable to the study of proteins. Although biophysical methods are capable of providing molecular-level details of protein–protein interactions, the increase in resolution to the molecular level is accompanied by an increased need to validate interpretation of the data. The chapter discusses the synergistic benefits of using multiple independent methods.