Most of the therapeutic antibodies approved for clinical use are full-size IgG1 molecules. The interaction of the IgG1 Fc with the neonatal Fc receptor (FcRn) plays a critical role in maintaining ...their long half-life. We have hypothesized that isolated Fc domains could be engineered to functionally mimic full-size IgG1 (nanoantibodies) but with decreased (10-fold) size. Here, we report for the first time the successful generation of a soluble, monomeric CH3 domain (mCH3). In contrast to the wild-type dimeric CH3, the mCH3 exhibited pH-dependent binding to FcRn similar to that of Fc. The binding free energy of mCH3 to FcRn was higher than that of isolated CH2 but lower than that of Fc. Therefore, CH3 may contribute a larger portion of the free energy of binding to FcRn than CH2. A fusion protein of mCH3 with an engineered antibody domain (m36.4) also bound to FcRn in a pH-dependent fashion and exhibited significantly higher neutralizing activity against HIV-1 than m36.4-Fc fusion proteins. The m36.4-mCH3 fusion protein was monomeric, stable, soluble, and expressed at a high level in Escherichia coli. We also found that engineering an additional disulfide bond in mCH3 remarkably increased its thermal stability, whereas the FcRn binding was not affected. These data suggest that mCH3 could not only help in the exploration of the dual mechanisms of the CH3 contribution to Fc functions (dimerization and FcRn interactions) but could also be used for the development of candidate therapeutics with optimized half-life, enhanced tissue penetration, access to sterically restricted binding sites, and increased therapeutic efficacy.
Solid-state nanopores within graphene-based materials are on the brink of fundamentally changing the sensing of desired bioanalytes through ion trafficking across nanoporous membranes. Here, we ...report on a two-electrode electrochemical biosensor comprised of a graphene oxide-polycarbonate track-etched nanosieve platform for the rapid and sensitive detection of the Human Immunodeficiency Virus Type 1 (HIV-1) envelope glycoprotein ectodomain (gp140MS). We have covalently linked an engineered high-affinity one-domain soluble CD4 fused to a human domain targeting HIV-1 coreceptor binding site and ferrocene (Fc) (2Dm2m) to the nanosieve platform. An exponential decrease in the ionic current resulted from a partial blockade of the nanosieve due to the specific interactions of gp140MS with the 2Dm2m protein, which was immobilized on the nanosieve platform by biolinkage as a function of applied voltages of 0.1–2.0 V. There was no change in current when a nonspecific antigen bovine serum albumin was tested under identical conditions. This platform had high sensitivity, and when the receptor-binding phenomenon was tested to identify the minimum concentration of target analyte, the lowest detection limit was as short as 8.3 fM and with sensitivity and response times of 0.87 mA mM–1 cm–1 and 12 s, respectively. In addition to this remarkable sensitivity, our nanobiorecognition platform has the advantage of superior stability due to the few layered graphene oxide laminates. It also exhibits exceptional biomolecule binding and higher reusability, sustainability, and ease of fabrication in a soft mechanism. Real samples of HIV positive and negative patients were successfully tested to confirm the virus by the developed platform. To the best of our knowledge, this is the first time prosperous pervious remembrance surface has been employed in a nanobiosensing application. In light of the recent great trend of using graphene-based nanopore surfaces created by sophisticated ion-beam methods in sensing and sequencing, this hybrid-surface nanolayer fabricated by the simple vacuum filtration of a few layered graphene oxide laminates may serve as a good alternative in terms of ease of fabrication without expensive instrumental prerequisites.
Soluble Monomeric IgG1 Fc Ying, Tianlei; Chen, Weizao; Gong, Rui ...
The Journal of biological chemistry,
06/2012, Letnik:
287, Številka:
23
Journal Article
Recenzirano
Odprti dostop
Antibody fragments are emerging as promising biopharmaceuticals because of their relatively small size and other unique properties. However, compared with full-size antibodies, these antibody ...fragments lack the ability to bind the neonatal Fc receptor (FcRn) and have reduced half-lives. Fc engineered to bind antigens but preserve interactions with FcRn and Fc fused with monomeric proteins currently are being developed as candidate therapeutics with prolonged half-lives; in these and other cases, Fc is a dimer of two CH2-CH3 chains. To further reduce the size of Fc but preserve FcRn binding, we generated three human soluble monomeric IgG1 Fcs (mFcs) by using a combination of structure-based rational protein design combined with multiple screening strategies. These mFcs were highly soluble and retained binding to human FcRn comparable with that of Fc. These results provide direct experimental evidence that efficient binding to human FcRn does not require human Fc dimerization. The newly identified mFcs are promising for the development of mFc fusion proteins and for novel types of mFc-based therapeutic antibodies of small size and long half-lives.
The Fc region of an antibody is a homodimer of two CH2-CH3 chains.
Monomeric IgG1 Fcs (mFcs) were generated by using a novel panning/screening procedure.
The mFcs are highly soluble and retain binding to human FcRn comparable with that of Fc.
The mFcs are promising for the development of novel therapeutic antibodies of small size and long half-lives.
Antibodies bound to human immunodeficiency virus type 1 (HIV-1) envelope protein expressed by infected cells mobilize antibody-dependent cellular cytotoxicity (ADCC) to eliminate the HIV-1-infected ...cells and thereby suppress HIV-1 infection and delay disease progression. Studies treating HIV-1-infected individuals with latency reactivation agents to reduce their latent HIV-1 reservoirs indicated that their HIV-1-specific immune responses were insufficient to effectively eliminate the reactivated latent HIV-1-infected T cells. Mobilization of ADCC may facilitate elimination of reactivated latent HIV-1-infected cells to deplete the HIV-1 reservoir and contribute to a functional HIV-1 cure. The most effective antibodies for controlling and eradicating HIV-1 infection would likely have the dual capacities of potently neutralizing a broad range of HIV-1 isolates and effectively mobilizing HIV-1-specific ADCC to eliminate HIV-1-infected cells. For this purpose, we constructed LSEVh-LS-F, a broadly neutralizing, defucosylated hexavalent fusion protein specific for both the CD4 and coreceptor gp120-binding sites. LSEVh-LS-F potently inhibited
HIV-1 and simian-human immunodeficiency virus (SHIV) infection in humanized mouse and macaque models, respectively, including
neutralization of HIV-1 strains resistant to the broadly neutralizing antibodies VRC01 and 3BNC117. We developed a novel humanized mouse model to evaluate
human NK cell-mediated elimination of HIV-1-infected cells by ADCC and utilized it to demonstrate that LSEVh-LS-F rapidly mobilized NK cells to eliminate >80% of HIV-1-infected cells
1 day after its administration. The capacity of LSEVh-LS-F to eliminate HIV-1-infected cells via ADCC combined with its broad neutralization activity supports its potential use as an immunotherapeutic agent to eliminate reactivated latent cells and deplete the HIV-1 reservoir.
Mobilization of antibody-dependent cellular cytotoxicity (ADCC) to eliminate reactivated latent HIV-1-infected cells is a strategy which may contribute to depleting the HIV-1 reservoir and achieving a functional HIV-1 cure. To more effectively mobilize ADCC, we designed and constructed LSEVh-LS-F, a broadly neutralizing, defucosylated hexavalent fusion protein specific for both the CD4 and coreceptor gp120-binding sites. LSEVh-LS-F potently inhibited
HIV-1 and SHIV infection in humanized mouse and macaque models, respectively, including
neutralization of an HIV-1 strain resistant to the broadly neutralizing antibodies VRC01 and 3BNC117. Using a novel humanized mouse model, we demonstrated that LSEVh-LS-F rapidly mobilized NK cells to eliminate >80% of HIV-1-infected cells
1 day after its administration. The capacity of LSEVh-LS-F to eliminate HIV-1-infected cells via ADCC combined with its broad neutralization activity supports its potential use as an immunotherapeutic agent to eliminate reactivated latent cells and deplete the HIV-1 reservoir.
Elicitation of antibodies with potent and broad neutralizing activity against HIV by immunization remains a challenge. Several monoclonal antibodies (mAbs) isolated from humans with HIV-1 infection ...exhibit such activity but vaccine immunogens based on structures containing their epitopes have not been successful for their elicitation. All known broadly neutralizing mAbs (bnmAbs) are immunoglobulin (Ig) Gs (IgGs) and highly somatically hypermutated which could impede their elicitation. Ig Ms (IgMs) are on average significantly less divergent from germline antibodies and are relevant for the development of vaccine immunogens but are underexplored compared to IgGs. Here we describe the identification and characterization of several human IgM-derived mAbs against HIV-1 which were selected from a large phage-displayed naive human antibody library constructed from blood, lymph nodes and spleens of 59 healthy donors. These antibodies bound with high affinity to recombinant envelope glycoproteins (gp140s, Envs) of HIV-1 isolates from different clades. They enhanced or did not neutralize infection by some of the HIV-1 primary isolates using CCR5 as a coreceptor but neutralized all CXCR4 isolates tested although weakly. One of these antibodies with relatively low degree of somatic hypermutation was more extensively characterized. It bound to a highly conserved region partially overlapping with the coreceptor binding site and close to but not overlapping with the CD4 binding site. These results suggest the existence of conserved structures that could direct the immune response to non-neutralizing or even enhancing antibodies which may represent a strategy used by the virus to escape neutralizing immune responses. Further studies will show whether such a strategy plays a role in HIV infection of humans, how important that role could be, and what the mechanisms of infection enhancement are. The newly identified mAbs could be used as reagents to further characterize conserved non-neutralizing, weakly neutralizing or enhancing epitopes and modify or remove them from candidate vaccine immunogens.
The currently available anti-HIV-1 drugs can control the infection but do not eradicate the virus. Their long-term use can lead to side effects and resistance to therapy. Therefore, eradication of ...the virus has been a major goal of research. Biological therapeutics including broadly neutralizing monoclonal antibodies (bnAbs) are promising tools to reach this goal. They could also help design novel vaccine immunogens potentially capable of eliciting bnAbs targeting the HIV-1 envelope glycoproteins (Envs).
We review HIV-1 bnAbs and their potential as candidate prophylactics and therapeutics used individually, in combination, or as bispecific fusion proteins. We also discuss their potential use in the 'activation-elimination' approach for HIV-1 eradication in infected patients receiving antiretroviral treatment as well as current vaccine design efforts based on understanding of interactions of candidate vaccine immunogens with matured bnAbs and their putative germline predecessors, and related antibody maturation pathways.
Exploration of HIV-1 bnAbs has provided and will continue to provide useful knowledge that helps develop novel types of biotherapeutics and vaccines. It is possible that bnAb-based candidate therapeutics could help eradicate HIV-1. Development of vaccine immunogens capable of eliciting potent bnAbs in humans remains a fundamental challenge.
Early human immunodeficiency virus type 1 (HIV-1) treatment during the acute period of infection can significantly limit the seeding of viral reservoirs and modify the course of disease. However, ...while a number of HIV-1 broadly neutralizing antibodies (bnAbs) have demonstrated remarkable efficacy as prophylaxis in macaques chronically infected with simian-human immunodeficiency virus (SHIV), intriguingly, their inhibitory effects were largely attenuated in the acute period of SHIV infection. To investigate the mechanism for the disparate performance of bnAbs in different periods of SHIV infection, we used LSEVh-LS-F, a bispecific bnAb targeting the CD4 binding site and CD4-induced epitopes, as a representative bnAb and assessed its potential therapeutic benefit in controlling virus replication in acutely or chronically SHIV-infected macaques. We found that a single infusion of LSEVh-LS-F resulted in rapid decline of plasma viral loads to undetectable levels without emergence of viral resistance in the chronically infected macaques. In contrast, the inhibitory effect was robust but transient in the acutely infected macaques, despite the fact that all macaques had comparable plasma viral loads initially. Infusing multiple doses of LSEVh-LS-F did not extend its inhibitory duration. Furthermore, the pharmacokinetics of the infused LSEVh-LS-F in the acutely SHIV-infected macaques significantly differed from that in the uninfected or chronically infected macaques. Host SHIV-specific immune responses may play a role in the viremia-dependent pharmacokinetics. Our results highlight the correlation between the fast clearance of infused bnAbs and the treatment failure in the acute period of SHIV infection and may have important implications for the therapeutic use of bnAbs to treat acute HIV infections.
Currently, there is no bnAb-based monotherapy that has been reported to clear the virus in the acute SHIV infection period. Since early HIV treatment is considered critical to restricting the establishment of viral reservoirs, investigation into the mechanism for treatment failure in acutely infected macaques would be important for the therapeutic use of bnAbs and eventually towards the functional cure of HIV/AIDS. Here we report the comparative study of the therapeutic efficacy of a bnAb in acutely and chronically SHIV-infected macaques. This study revealed the correlation between the fast clearance of infused bnAbs and treatment failure during the acute period of infection.
PDF
