Gerald R. Nakamura

Delineation of a region of the human immunodeficiency virus type 1 gp120 glycoprotein critical for interaction with the CD4 receptor

The primary event in the infection of cells by HIV is the interaction between the viral envelope glycoprotein, gp120, and its cellular receptor, CD4. A recombinant form of gp120 was found to bind to a recombinant CD4 antigen with high affinity. Two gp120-specific murine monoclonal antibodies were able to block the interaction between gp120 and CD4. The gp120 epitope of one of these antibodies was isolated by immunoaffinity chromatography of acid-cleaved gp120 and shown to be contained within amino acids 397-439. Using in vitro mutagenesis, we have found that deletion of 12 amino acids from this region of gp120 leads to a complete loss of binding. In addition, a single amino acid substitution in this region results in significantly decreased binding, suggesting that sequences within this region are directly involved in the binding of gp120 to the CD4 receptor.

Genetic and Immunologic Characterization of Viruses Infecting MN-rgp120-Vaccinated Volunteers

Proviral sequences were determined and immunologic characterization was carried out for envelope glycoproteins from 7 vaccinees who became infected with human immunodeficiency virus type 1 (HIV-1), through high-risk behavior, while participating in clinical trials of MN-rgp120, a candidate HIV-1 vaccine. All 7 infections resulted from subtype B viruses; however, only 3 of the viruses possessed the MN serotype-defining V3 domain sequence, IGPGRAF, prevalent in 60%-70% of US infections. Six of the 7 viruses differed from MN-rgp120 at a neutralizing epitope in the C4 domain, and all 7 differed from MN-rgp120 at a neutralizing epitope in the V2 domain. Recombinant gp120 was prepared from each breakthrough specimen and tested for binding to a panel of neutralizing monoclonal antibodies. The results suggest that 6 of 7 breakthrough infections may be related to incomplete immunization or to infection with viruses that differed from the vaccine immunogen at important virus-neutralizing epitopes.

Rapid Identification of Small Binding Motifs with High-Throughput Phage Display: Discovery of Peptidic Antagonists of IGF-1 Function

A panel of 22 naïve peptide libraries was constructed in a polyvalent phage display format and sorted against insulin-like growth factor-1 (IGF-1). The libraries were pooled to achieve a total diversity of 4.4 x 10(11). After three rounds of selection, the majority of the phage clones bound specifically to IGF-1, with a disulfide-constrained CX(9)C scaffold dominating the selection. Four monovalently displayed sub-libraries were designed on the basis of these conserved motifs. Sub-library maturation in a monovalent format yielded an antagonistic peptide that inhibited the interactions between IGF-1 and two cell-surface receptors and those between IGF-1 and two soluble IGF binding proteins with micromolar potency. NMR analysis revealed that the peptide is highly structured in the absence of IGF-1, and peptides that preorganize the binding elements were selected during the sorting.

Structural Basis of Signaling Blockade by Anti-IL-13 Antibody Lebrikizumab.

The cytokine interleukin 13 (IL-13) is a major effector molecule for T-helper type 2 inflammation and is pathogenic in allergic diseases such as asthma. The effects of IL-13 are mediated via a pathway that is initiated by binding to a heterodimeric receptor consisting of IL-13Rα1 and IL-4Rα. Antibodies raised against IL-13 can block its inflammatory effects by interfering with binding to either of the two receptor polypeptides. Lebrikizumab is a monoclonal anti-IL-13 antibody that has shown clinical benefit in a phase II study for the treatment of moderate-to-severe uncontrolled asthma. Here we report the molecular structure of IL-13 in complex with the Fab from lebrikizumab by X-ray crystallography at 1.9Å resolution. We show that lebrikizumab inhibits IL-13 signaling by binding to IL-13 with very high affinity and blocking IL-13 binding to IL-4Rα. In addition, we use site-directed mutations to identify the most important antibody contributors to binding. Our studies define key features of lebrikizumab binding and its mechanism of action that may contribute to its clinical effects.

Stable "zeta" peptides that act as potent antagonists of the high-affinity IgE receptor.

Recently we described a family of peptides, unrelated in sequence to IgE, that form stable β-hairpins in solution and inhibit IgE activity in the μM range [Nakamura, G. R., Starovasnik, M. A., Reynolds, M. E. & Lowman, H. B. (2001) Biochemistry 40, 9828–9835]. Using an expanded set of peptide–phage libraries, we found a simpler motif, X2CPX2CYX, for binding to the high-affinity IgE receptor. In solution, one of these peptides spontaneously formed a covalent antiparallel dimer. We subsequently linked these monomers in a single-chain construct on phage and optimized receptor binding. Ultimately, peptides with 30 nM affinity were produced. NMR studies showed that the peptide adopts a stable fold consisting of two “zeta” (ζ)-shaped moieties. Structure–activity analyses reveal a single binding site created by the zeta-dimer, with two tyrosine residues important for structural stability and two proline residues important for FcɛRI binding. The peptides inhibit histamine release from cultured cells and are extremely stable in biological fluids. The zeta peptides appear to act as competitive IgE inhibitors and suggest possibilities for design of novel IgE antagonists.

Development of a human IgG4 bispecific antibody for dual targeting of interleukin-4 (IL-4) and interleukin-13 (IL-13) cytokines

Background: Dual neutralization of IL-4 and IL-13 is a promising therapeutic approach for asthma and allergy. Results: Knobs-into-holes IgG1 and IgG4 bispecific antibodies targeting both cytokines were developed. Conclusion: Bispecific antibodies of both isotypes have comparable in vitro potencies, in vivo pharmacokinetics, and lung partitioning. Significance: Further extension of knobs-into-holes technology to human IgG4 isotype as reported here provides greater options for therapeutics. Human bispecific antibodies have great potential for the treatment of human diseases. Although human IgG1 bispecific antibodies have been generated, few attempts have been reported in the scientific literature that extend bispecific antibodies to other human antibody isotypes. In this paper, we report our work expanding the knobs-into-holes bispecific antibody technology to the human IgG4 isotype. We apply this approach to generate a bispecific antibody that targets IL-4 and IL-13, two cytokines that play roles in type 2 inflammation. We show that IgG4 bispecific antibodies can be generated in large quantities with equivalent efficiency and quality and have comparable pharmacokinetic properties and lung partitioning, compared with the IgG1 isotype. This work broadens the range of published therapeutic bispecific antibodies with natural surface architecture and provides additional options for the generation of bispecific antibodies with differing effector functions through the use of different antibody isotypes.

Comparison of the immune response to recombinant gp120 in humans and chimpanzees

ObjectiveTo assess similarities and differences in antibody responses to recombinant (r) HIV-1IIIMB gp120 in chimpanzees, previously protected from HIV-1 infection, and human volunteers immunized in connection with a Phase I clinical trial. MethodsFrozen sera from humans immunized with rgp120 from HIV-IIIB and chimpanzees immunized with the same antigen or recombinant soluble gp160 were compared in a variety of serologic assays. ResultsThe magnitude of the antibody response to gp120 was similar in both species; however, the half-life of the antibody response to rgp120 was approximately 4.5 times longer in humans (9 weeks) than in chimpanzees (2 weeks). Antibodies to gp120 in both species were broadly cross-reactive with gp120 from diverse isolates of HIV-1 and were effective in blocking the binding of gp120 to CD4. Antibody binding to native gp120 was greater than to denatured gp120 in both species. Antibody responses to the principal neutralizing determinant (V3 domain) and virus neutralization titers were approximately 10-fold lower in humans than chimpanzees. The relative avidity of antibody binding to gp120 was higher in the sera from the immunized chimpanzees than in the immunized humans. ConclusionsWhile the antibody responses to rgp120 elicited in man and chimpanzees were in many ways similar, significant differences did occur. Predictions made on the basis of chimpanzee immunogenicity studies overestimated the potency of the virus neutralizing titers and under-estimated the duration of the antibody response achieved in humans.

Monoclonal Antibodies to the V2 Domain of MN-rgp120: Fine Mapping of Epitopes and Inhibition of α4β7 Binding

Background Recombinant gp120 (MN-rgp120) was a major component of the AIDSVAX B/E vaccine used in the RV144 trial. This was the first clinical trial to show that vaccination could prevent HIV infection in humans. A recent RV144 correlates of protection study found that protection correlated with the presence of antibodies to the V2 domain. It has been proposed that antibodies to the α4β7 binding site in the V2 domain might prevent HIV-1 infection by blocking the ability of virions to recognize α4β7 on activated T-cells. In this study we investigated the specificity of monoclonal antibodies (MAbs) to the V2 domain of MN-rgp120 and examined the possibility that these antibodies could inhibit the binding of MN-rgp120 to the α4β7 integrin. Methodology/Principal Findings Nine MAbs to the V2 domain were isolated from mice immunized with recombinant envelope proteins. The ability of these MAbs to inhibit HIV infection, block the binding of gp120 to CD4, and block the binding of MN-rgp120 to the α4β7 integrin was measured. Mutational analysis showed that eight of the MAbs recognized two immunodominant clusters of amino acids (166–168 and 178–183) located at either end of the C strand within the four-strand anti-parallel sheet structure comprising the V1/V2 domain. Conclusions/Significance These studies showed that the antigenic structure of the V2 domain is exceedingly complex and that MAbs isolated from mice immunized with MN-rgp120 exhibited a high level of strain specificity compared to MAbs to the V2 domain isolated from HIV-infected humans. We found that immunization with MN-rgp120 readily elicits antibodies to the V2 domain and some of these were able to block the binding of MN-rgp120 to the α4β7 integrin.

A Neutralizing Anti-gH/gL Monoclonal Antibody Is Protective in the Guinea Pig Model of Congenital CMV Infection

Human cytomegalovirus (HCMV) is the most common cause of congenital virus infection. Congenital HCMV infection occurs in 0.2–1% of all births, and causes birth defects and developmental abnormalities, including sensorineural hearing loss and developmental delay. Several key studies have established the guinea pig as a tractable model for the study of congenital HCMV infection and have shown that polyclonal antibodies can be protective [1]–[3]. In this study, we demonstrate that an anti-guinea pig CMV (GPCMV) glycoprotein H/glycoprotein L neutralizing monoclonal antibody protects against fetal infection and loss in the guinea pig. Furthermore, we have delineated the kinetics of GPCMV congenital infection, from maternal infection (salivary glands, seroconversion, placenta) to fetal infection (fetus and amniotic fluid). Our studies support the hypothesis that a neutralizing monoclonal antibody targeting an envelope GPCMV glycoprotein can protect the fetus from infection and may shed light on the therapeutic intervention of HCMV congenital infection in humans.

Binding Protein-3-Selective Insulin-Like Growth Factor I Variants: Engineering, Biodistributions, and Clearance

Insulin-like growth factor I (IGF-I) is a potent anabolic peptide that mediates most of its pleiotropic effects through association with the IGF type I receptor. Biological availability and plasma half-life of IGF-I are modulated by soluble binding proteins (IGFBPs), which sequester free IGF-I into high affinity complexes. Elevated levels of specific IGFBPs have been observed in several pathological conditions, resulting in inhibition of IGF-I activity. Administration of IGF-I variants that are unable to bind to the up-regulated IGFBP species could potentially counteract this effect. We engineered two IGFBP-selective variants that demonstrated 700- and 80,000-fold apparent reductions in affinity for IGFBP-1 while preserving low nanomolar affinity for IGFBP-3, the major carrier of IGF-I in plasma. Both variants displayed wild-type-like potency in cellular receptor kinase assays, stimulated human cartilage matrix synthesis, and retained their ability to associate with the acid-labile subunit in complex with IGFBP-3. Furthermore, pharmacokinetic parameters and tissue distribution of the IGF-I variants in rats differed from those of wild-type IGF-I as a function of their IGFBP affinities. These IGF-I variants may potentially be useful for treating disease conditions associated with up-regulated IGFBP-1 levels, such as chronic or acute renal and hepatic failure or uncontrolled diabetes. More generally, these results suggest that the complex biology of IGF-I may be clarified through in vivo studies of IGFBP-selective variants.