JAIDS Journal of Acquired Immune Deficiency Syndromes:
Basic and Translational Science
Rational Design and Characterization of the Novel, Broad and Potent Bispecific HIV-1 Neutralizing Antibody iMabm36
Sun, Ming BSc*,†; Pace, Craig S. PhD*; Yao, Xin PhD*; Yu, Faye BSc*; Padte, Neal N. PhD*; Huang, Yaoxing PhD*; Seaman, Michael S. PhD‡; Li, Qihan MD†; Ho, David D. MD*,†
*Aaron Diamond AIDS Research Center, The Rockefeller University, New York, NY;
†Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College, Kunming, China; and
‡Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA.
Correspondence to: David D. Ho, MD, The Aaron Diamond AIDS Research Center, New York, NY (e-mail: firstname.lastname@example.org and Qihan Li, MD, Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College, Kunming, China (e-mail: email@example.com).
D.D.H. was supported by the Bill and Melinda Gates Foundation's Collaboration for AIDS Vaccine Discovery (CAVD), grant numbers OPP50714 and OPP1040731, and by the National Institutes of Health (NIH) grant number 1DP1DA033263-01. M.S. was supported by the Bill and Melinda Gates Foundation's Comprehensive Antibody Vaccine Immune Monitoring Consortium (CA-VIMC), grant number 1032144.
The authors have no conflicts of interest to disclose.
Received April 16, 2014
Accepted May 07, 2014
Background: Although broadly neutralizing monoclonal antibodies (bNAbs) have always been considered to be a potential therapeutic option for the prophylaxis and treatment of HIV infection, their lack of breadth against all HIV variants has been one of the limiting factors. To provide sufficient neutralization breadth and potency against diverse viruses, including neutralization escape mutants, strategies to combine different bNAbs have been explored recently.
Methods: We rationally designed and engineered a novel bispecific HIV-1–neutralizing antibody (bibNAb), iMabm36. The potency and breadth of iMabm36 against HIV were extensively characterized in vitro.
Results: iMabm36 comprises the anti-CD4 Ab ibalizumab (iMab) linked to 2 copies of the single-domain Ab m36, which targets a highly conserved CD4-induced epitope. iMabm36 neutralizes a majority of a large, multiclade panel of pseudoviruses (96%, n = 118) at an IC50 concentration of less than 10 µg/mL, with 83% neutralized at an IC50 concentration of less than 0.1 µg/mL. In addition, iMabm36 neutralizes a small panel of replication-competent transmitted-founder viruses to 100% inhibition at a concentration of less than 0.1 µg/mL in a peripheral blood mononuclear cell–based neutralizing assay. Mechanistically, the improved antiviral activity of iMabm36 is dependent on both the CD4-binding activity of the iMab component and the CD4i-binding activity of the m36 component. After characterizing that viral resistance to iMabm36 neutralization was due to mutations residing in the bridging sheet of gp120, an optimized m36 variant was engineered that, when fused to iMab, improved antiviral activity significantly.
Conclusions: The interdependency of this dual mechanism of action enables iMabm36 to potently inhibit HIV-1 entry. These results demonstrate that mechanistic-based design of bibNAbs can generate potential preventive and therapeutic candidates for HIV/AIDS.
Broad and potent antibodies represent a new generation of antiviral agents for the prophylaxis and treatment of HIV infection.1 Compared with the small molecule antiretrovirals currently used to treat HIV, antibodies are generally considered safer and have longer half-lives, and have been shown to provide passive protection against mucosal challenge in the Simian/Human Immunodeficiency Virus (SHIV) macaque model. These promising features, combined with the greater breadth and potency of the newest generation of antibodies, have reignited the interest in developing antibody-based drugs against HIV-1. Recently, through microneutralization screening of B-cell cultures and single B-cell sorting from HIV-1–infected patients, several researchers have successfully isolated and characterized many new monoclonal antibodies, such as PG9/16, VRC01, 3BNC117, NIH45-46, the PGT Abs, and 10E8.2–6 Several broadly neutralizing mAbs (bNAbs), when administered as monotherapy, can protect against HIV-1 infection in animal models.7,8 However, their efficacy in treating an established infection is limited.8–11 In particular, viral rebound occurs quickly in all patients receiving bNAbs due to the outgrowth of preexisting or de novo viral escape variants.12 It has been reported that combining multiple neutralizing antibodies that each use a different mechanism of action would increase the antiviral potency and barrier to resistance in vivo than any one antibody alone.8,10,11,13–15 Therefore, novel antibodies with a broader neutralizing activity and greater potency are needed in the defense of HIV-1 resistance and resistance development.
HIV-1 entry is triggered by the interaction of the viral envelope (Env) glycoprotein gp120 with domain 1 (D1) of the T-cell coreceptor CD4.16,17 Binding of CD4 by gp120 induces extensive conformational changes in gp120 leading to the formation and exposure of the coreceptor (CoR)-binding site, also known as the CD4-induced (CD4i) site, on gp120.18–20 The CoR-binding site is typically unformed on native Env trimers before CD4 engagement. The CoR-binding site is highly immunogenic and elicits a class of Abs known as CD4i Abs in vivo. The bridging sheet of gp120 is a critical component of the CoR-binding site that is conserved across genetically diverse HIV-1 isolates from different clades.21,22 However, access of full-size Abs to the CD4i epitope (bridging sheet) is sterically restricted during viral entry into cells, most likely because the large size of a full length Ab cannot access the tight crypt within the envelope where the bridging sheet resides.23,24 Thus, most known full-size CD4i Abs do not have potent antiviral activity. Fragments of CD4i Abs that are smaller in size could potentially gain access to the CD4i epitope during viral entry and have been shown to inhibit HIV entry more potently than full-size Abs.25 m36, a single-domain Ab that is 15 kDa in size, was isolated from a naive human Ab library and targets the highly conserved, but sterically restricted, CD4i epitope on HIV Env.26,27 m36 has been reported to be one of the most potent and broadly crossreactive HIV-1 engineered antibody domains with a mean IC50 in the 100-nM range.26,27 However, similar to other antibody fragments, the m36 polypeptide is predicted to have a short half-life in circulation due to its relatively small size.26,28
Ibalizumab (iMab) is an mAb that has a broad and potent activity against HIV-1.29−31 It inhibits HIV by binding mainly to domain 2 (D2) of the CD4 on host target cells, inhibiting post-CD4 binding events required to infect cells.32 In a large panel of diverse, clinically relevant HIV-1 pseudoviruses (n = 118), iMab neutralized 92% of viruses, as defined by 50% inhibition of infection, and 47% of viruses, as defined by 90% inhibition of infection.31
We have previously demonstrated that iMab-based bispecific bNabs exhibit synergistic antiviral activity compared with the parental Abs, either alone or in combination, attributed in part to the enhanced local concentration of bNAb activity at the site of viral entry.31 Fine epitope mapping and crystal structure resolution of the iMab–CD4 interaction have previously shown that the iMab does not interfere with the binding of gp120 to CD4,32−34 and preliminary data from our laboratory suggest that iMab does not impair exposure and engagement of the HIV-1 bridging sheet with the HIV-1 coreceptors. Thus, we hypothesized that fusing a CD4i Ab to iMab would anchor the activity of CD4i Abs at the virological synapse before gp120–CD4 engagement, thereby diminishing the spatial constraints that impair native CD4i Abs from accessing their epitope and allowing the bispecific Ab to potently inhibit HIV-1 entry at 2 distinct, but spatially related, entry steps. To test this, we engineered iMabm36, a novel bispecific antibody that could target both these entry steps simultaneously. iMabm36 comprised the anti-CD4 Ab iMab linked to 2 copies of the anti-CD4i, single-domain Ab m36. We show that iMabm36 potently inhibits the viral entry of many iMab-resistant viruses without affecting the inhibition of iMab-sensitive viruses. Also, fusion of m36 to iMab is predicted to extend the short life of m36 in vivo. Thus, the novel, rationally designed bispecific HIV-1 neutralizing antibody (bibNab) iMabm36 provides enhanced anti-HIV-1 activity compared with either parental Ab alone, or in combination, and may be a valuable new therapeutic for the prevention or treatment of HIV-1 infection.
Recombinant soluble CD4 protein and TZM-bl cells were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. iMab was provided by TaiMed Biologics USA.31,35
Construction and Expression of iMabm36 Fusion Ab
The following primers were used:
A bispecific fusion Ab was constructed based on m36 and a derivative IgG1 version of iMab. As shown in Figure 1, m36 was linked to the C-terminus of the heavy chain of iMab via a flexible (G4S)3 linker peptide (GGGGSGGGGSGGGGSG). The cDNA sequence of the fusion construct was generated by overlap polymerase chain reaction (PCR) using primersm36F1, m36F2, m36R1, and m36R2. Subsequently, the products were digested with NheI and XhoI and cloned into the pVAX expression plasmid (Life Technologies, Grand Island, NY). PRO140m36, (G4S)1 linked m36, and (G4S)5 linked m36 were cloned into pVAX in a similar manner. To generate ΔiMabm36, the ΔiMabm36 gene was amplified by mutagenesis PCR primers (iMab Δ1 and iMab Δ2) using the iMabm36-encoding plasmid as a template. Similarly, the iMabΔm36 gene was obtained by mutagenesis PCR by primers (m36Δ1F, m36 Δ1R, m36 Δ2F, m36 Δ2R, and m36 Δ3). m36 (CDR3 E51) fragments were amplified by overlap PCR and cloned into a pVAX expression plasmid. All constructs were confirmed by direct nucleotide sequencing. Plasmid DNA was isolated by anion exchange using endotoxin-free Maxi kits (Qiagen, Valencia, CA). For expression of different Abs, 293 A cells were transiently cotransfected by polyethylenimine at a final concentration of 5 μg/mL and 10 μg of pVAX vectors expressing the heavy chain iMabm36 fusion and the light chain of iMab. After 72 hours, cell culture supernatants were harvested and analyzed for the presence of antibody by capture enzyme-linked immunosorbent assay (ELISA). Ab-containing culture supernatants were filtered and purified by affinity chromatography using a ProteinA column (Pierce, Rockford, IL) and concentrated with a centrifugal filter (Millipore, Billerica, MA).
In Vitro Structural Integrity of iMabm36
Rabbits were immunized with purified m36 protein (300 μg per dose) in Complete Freund's Adjuvant (CFA) at week 0 and subsequently boosted in Incomplete Freund's Adjuvant (IFA) twice at weeks 4 and 8. Anti-m36 Ab titers were determined in the serum sample collected 4 weeks after the last boost immunization. The in vitro integrity of the iMabm36 fusion Ab was determined by incubation of the fusion Ab in 20% mouse serum in phosphate-buffered saline at 37°C for up to 7 days. Aliquots of the untreated (day 0) and treated fusion Ab were taken at the indicated time points and stored at −20°C. The presence of intact iMabm36 was examined by the functional binding activity of iMabm36 to sCD4 and determined by an anti-iMab Fc direct ELISA and anti-m36 sandwich ELISA, respectively. The presence of functional, intact iMabm36 was also assessed for antiviral activity by the TZM-bl neutralization assay.
Purified iMabm36 Was Assessed for Soluble CD4 (sCD4) Binding in a Competition ELISA Assay
Soluble hCD4 was adsorbed onto 96-well (0.1 μg per well) high-binding ELISA plates (Costar/Corning, Corning, NY). The plates were then blocked with 4% dehydrated milk and 1% bovine serum albumin in phosphate-buffered saline–Tween (blocking buffer). The plates were washed and a fixed concentration of horse radish peroxidase (HRP)-labeled iMab (2.5 μg/mL) was then mixed with increasing concentrations of iMabm36 or unlabeled iMab and measured for sCD4-binding competition. Plates were then washed, developed by means of a streptavidin-coupled peroxidase and tetramethylbenzidine (TMB) substrate (Sigma, St. Louis, MO), and measured on an ELISA plate reader at an optical density of 450 nm.
Pseudovirus Preparation and Generation of Bridging Sheet Mutants
The following primers were used to generate bridging sheet mutant viruses:
Assay stocks of molecularly cloned Env-pseudotyped viruses were prepared by cotransfecting 293T cells with an Env-expressing plasmid and an Env-deficient backbone plasmid (SG3ΔEnv) at a ratio of 1:3 using polyethylenimine. All viral supernatants were harvested 2 days after transfection, and the TCID50 was determined on TZM-bl cells by end-point dilution. Q23.17, Q259.d2.17, and T28-50 Env-expressing plasmids coding bridging sheet residues were substituted with clade B consensus amino acids by site-directed mutagenesis according to the manufacturer's instructions (Agilent, Santa Clara, CA). Six Env-pseudotyped mutants (Q23-β21mut, Q23-β3β21mut, Q259-β21mut, Q259-β3β21mut, T278-β21mut, and T278-β3β21mut) were also prepared as described above.
Table. No title avai...Image Tools
Pseudovirion-Based and Peripheral Blood Mononuclear Cell–Based Neutralization Assay
Neutralization against Env pseudoviruses was measured with a luciferase-based assay in TZM-bl cells as previously described.36 Briefly, 5-fold serial dilutions of antibodies were performed in triplicate (96-well flat bottom plate) in TZM-bl cells (1 × 104 per well in a 100-μL volume) and incubated for 1 hour at 37°C. The 100 50% Tissue Culture Infective Dose (TCID50) of virus and 50 μL of 10% Dulbecco modified Eagle medium (a growth medium), containing diethylaminoethyl cellulose–dextran (Sigma, St. Louis, MO) at a final concentration of 11 μg/mL were added to each well. Assay controls included TZM-bl cells alone (cell control) and TZM-bl cells with virus (virus control). After a 48-hour incubation at 37°C, the assay medium was removed from each well, and 40 μL of lysis buffer and 60 μL Galacto-Star luciferase reagents (Applied Biosystems, Foster City, CA) were added, and luminescence was measured. The IC50 titer was calculated as the antibody dilution that caused a 50% reduction in relative luminescence units compared with that for the virus control after subtraction of cell control relative luminescence units.
A standard peripheral blood mononuclear cell–based neutralization assay was used to assess iMabm36 antiviral activity. Briefly, the neutralizing assay was performed in a 96-well plate format. Phytohemagglutinin and Interleukin 2- (PHA/IL2-) activated PBMCs (1.5 × 105 per well) were infected with virus in the presence or absence of antibody. The PBMCs were washed extensively after overnight culture. Culture supernatants were collected on days 3 and 7. Viral p24-antigen was measured by means of a commercial ELISA (Beckman Coulter, Brea, CA).
iMabm36 Construction and Characterization
To generate iMabm36, we fused m36 to the C-terminus of the heavy chain of iMab via a flexible (G4S)3 linker peptide (Fig. 1A). The iMab, m36, and iMabm36 expression cassettes correctly produced Ab molecules at the predicted molecular weights (Fig. 1B). To confirm whether the CD4-binding activity of iMabm36 was unaltered compared with parental iMab, we evaluated its sCD4 binding activity in vitro. Indeed, iMabm36 was able to compete with HRP-labeled iMab for sCD4 binding in vitro with equal potency as that of the parental unlabeled iMab, indicating that the fusion of m36 to iMab did not impair its CD4-binding function. As expected, m36 alone did not compete with iMab binding to sCD4 (Fig. 1C).
To assess the structural integrity and stability of iMabm36, we first generated high titers of rabbit anti-m36 immune serum. The integrity of iMabm36 was examined by a secondary Ab against iMab Fc or anti-m36 rabbit immune serum upon overnight incubation of iMabm36 at 37°C. No loss of CD4− or m36-binding was observed. In addition, no loss of neutralization activity was observed from samples incubated at 37°C for 7 days (data not shown). Thus, the functional activity of iMabm36 is retained for up to at least 7 days in the conditions tested.
iMabm36 Fusion Ab Improves Antiviral Breadth Compared With iMab or m36 Alone
To examine if the fusion of m36 to the C-terminal of iMab could result in more potent antiviral activity, we first tested iMabm36, iMab, and m36 in a TZM-bl neutralization assay against a panel of 6 viruses that included iMab-sensitive and resistant viruses. For all viruses tested, the potency of iMab36 was enhanced as compared with one of the parental components, m36, alone. For iMab-sensitive viruses specifically (CQLDR03-A2 and SC20 8A8A), the potencies of iMabm36 and iMab were similar (Fig. 2A). However, iMabm36 potently neutralized all 4 iMab-resistant viruses (9077.12 B5A, TT31P 2F10, SC33 4H1, and RHPA4259.1mut)31 better than either iMab or m36 alone did, and achieved a 100% maximum percent inhibition (MPI) at low nanomolar concentrations (Fig. 2B). Consistent with what was previously reported, the 50% inhibitory concentration of m36 is normally within the range of hundreds of nanomolar concentrations.26 The neutralizing activity of iMabm36 was also assessed in a PBMC-based 7-day neutralizing assay against 6 replication-competent transmitted-founder viruses. Both iMab and iMabm36 achieved 100% neutralization against this panel of viruses. However, iMabm36 seemed to improve antiviral potency in this assay, with 100% inhibition at a mean concentration of 0.1 μg/mL (Fig. 2C).
Increased Antiviral Potency of iMabm36 Compared With the iMab and m36 Mixture
The results above indicated that the combination of iMabm36 is more active than its respective individual components iMab and m36. To investigate if the greater antiviral activity of iMabm36 could also be achieved by simply mixing iMab and m36, we performed a neutralizing assay using iMab and m36 at a molar ratio of 1:2 because one iMabm36 molecule carries 2 m36 domains. Mixing of the individual parental components, iMab and m36, indeed improved the antiviral activity as compared with either of the parental components alone, achieving 100% neutralization of iMab-resistant viruses (RHPA4259.1mut, 9077.12 B5A, and TT31P 2F10) (Fig. 3A). However, iMabm36 bispecific antibody had an even greater antiviral activity, being at least 10-fold more potent than the coadministration of iMab and m36 parental components.
To test if iMabm36 acts by increasing the local concentration of m36 at the cell surface, we compared the antiviral activity of mixing iMab with m36 at a molar ratio of 1:10 against the same small panel of viruses (Fig.3B). A mixture of iMab and m36 at a 1:10 ratio was more potent than a mixture of iMab and m36 at a 1:2 ratio. Notably, even with a 5-fold excess of m36, the antibody mixture was still less potent than the bispecific molecule. These results suggest that iMab and m36, once fused into a single antibody-like molecule, contribute to the antiviral activity in a synergistic manner.
Improved Antiviral Potency of iMabm36 Is Dependent on Its CD4-Binding Activity and Sensitivity to m36
To understand the mechanism of the improved antiviral activity of the iMab36 fusion, we ablated its ability to bind CD4 by mutating 2 contact residues, Glu (E) and Try (Y), in the iMab CDR H3 to alanine, yielding ΔiMabm36 (Fig. 4A, left panel).33 An ELISA was conducted to confirm that ΔiMabm36 lost its capability to compete with iMab for CD4 binding (Fig. 4A, right panel). Correspondingly, ΔiMabm36 also lost its neutralization activity against the viruses tested (CQLDR03-A2 and RHPA4259.1mut) (Fig. 4B), indicating the CD4-binding activity of iMabm36 is critical for its activity.
Because the gp120-bridging sheet interacts directly with the HIV-1 coreceptors, we assessed whether the activity of iMabm36 was dependent specifically on the CD4-binding activity, or if anchoring m36 to CCR5 via the anti-CCR5 mAb PRO140 could also augment the activity of m36. Thus, we replaced the iMab component of iMabm36 with PRO14037 to generate PRO140m36 and assessed its activity against the prototypic CXCR4-tropic virus, NL4-3, which is therefore resistant to PRO140. Here, PRO140m36 was indeed approximately 10-fold more potent than m36, indicating that both CD4-anchoring and CCR5-anchoring of m36 can augment the neutralization activity of m36 (Fig. 4C).
To assess the contribution of m36 specificity in the context of iMabm36, we replaced all acidic and tyrosine residues of the m36 CDRs with alanine, because acidic and tyrosine residues are considered vital to the activity of CD4i Abs.21,35,38 These substitutions in iMabΔm36 abolished the improved neutralization activity of iMabm36 such that its neutralization activity closely resembled that of iMab (Fig. 4D). These results indicate that the antiviral activity of iMabm36 is dependent on both its CD4 binding activity and its intrinsic m36 activity. Further, in analyzing the neutralization data against 9 iMab-resistant viruses, we noticed a highly significant direct correlation (r2 = 0.93, P < 0.001) of the IC50 of m36 and the IC50 of iMabm36 (Fig. 4E). Together, these results suggest that, in the context of iMab-resistant viruses, the potency of iMabm36 is determined by the virus sensitivity to m36.
To more comprehensively assess its breadth and potency against HIV-1, iMabm36 activity was further subjected to testing against a large panel of HIV-1 pseudoviruses (n = 118) representing all major circulating HIV-1 subtypes. Although iMabm36 did not exhibit a significantly improved breadth of neutralization compared with that of iMab (P = 0.4), neutralizing 96% and 92% of the panel, respectively, iMabm36 was significantly more effective at inhibiting HIV-1 infection, achieving on average 91.5% MPI compared with only 81.8% by iMab (P < 0.001). A majority (83%) of this panel of viruses were neutralized by iMabm36 at an IC50 of <0.1 μg/mL compared with 75% for iMab (Fig. 5A). iMabm36 exhibited IC80 values of <7 nM (<1 μg/mL) for 45.7% of the viruses tested, compared with 40.7% for iMab (Fig. 5B). These results suggest that the fusion of m36 to iMab (iMabm36) improves the antiviral activity of iMab.
Understanding iMabm36 Resistance
In analyzing the neutralization data from the large panel of HIV-1 pseudoviruses (n = 118), m36 fusion to iMab improved the neutralization activity of iMab against the viruses of all clades except for clades A and G (Fig. 6A). To assess whether clade A viruses were resistant to iMabm36 because they lack the m36 epitope, we aligned and compared the sequences of their gp120 bridging sheets (β3, β2, β21, and β20 strands) that contain the putative epitope of m36 to the consensus B sequence.27 Based on the comparison, we substituted specific residues in the β3 and β21 sheets of Q23.17 (clade A), Q259.d2.17 (clade A), and T28-50 (clade AG) Env with clade B consensus amino acids (Table 1), because m36 was initially isolated by phage display using a clade B gp120 as bait.26,27 As shown in Figure 6B, the resistant viruses with β21 site-directed mutations to clade B consensus residues were more sensitive to iMabm36. Further, the combination of the β3β21 mutations rendered the viruses highly sensitive to iMabm36, indicating that bridging sheet sequence divergence of clade A/AG viruses confers iMabm36 resistance.
Modifications to Improve iMabm36 Antiviral Activity
To investigate the impact of the linker length on the antiviral activity of iMabm36, we compared the neutralization activity of the original iMabm36 (G4S)3 to iMabm36 with a short linker (G4S)1 and iMabm36 with a long linker (G4S)5. iMabm36 (G4S)3 had a greater antiviral activity than iMabm36 with a short linker (G4S)1. iMabm36 with a long linker (G4S)5 was more active against iMabm36 resistant viruses 1006_11_C3_1601 and du172.17 (Fig. 7A). Thus, a longer linker between iMab and m36 improves the antiviral activity of the fusion Ab.
To improve iMabm36 activity against clade A viruses, we modified the m36 CDRs. In particular, we substituted m36 CDR H3 with that of the CDR H3 from the CD4i Ab E51, because this CDR has more favorable electrostatic interactions and a greater binding interface with gp120 than m36.39,40 When tested against iMabm36-resistant pseudoviruses (Q23.17 and Q259.d2.17), the resultant modified iMabm36 (CDR3 E51) exhibited more potent neutralizing activity against these 2 viruses tested (Fig. 7B).
Finally, we combined the CDR H3 modification [m36(CDR3 E51)] with iMabm36L5 [the iMabm36 variant with the longer linker (G4S)5] to produce an optimized iMabm36, termed iMabm36opt. When tested against iMabm36-sensitive and resistant pseudoviruses, the iMabm36opt retained its antiviral activity against iMabm36-sensitive viruses, and neutralized resistant viruses more effectively than did iMabm36, iMabm36L5, and iMabm36(CDR3 E51), achieving an MPI of 86%–98% compared with only 40%–87% for iMabm36, 65%–94% for iMabm36L5, and 75%–91% for iMabm36(CDR3 E51) (data not shown). On analyzing MPIs from 9 iMabm36-resistant viruses tested, a highly significant improvement (P = 0.0029) of the MPI for iMabm36opt was observed (Fig. 7C). Thus, these data suggest that the optimized variants of iMabm36 can exhibit greater potency antiviral activity than can the original iMabm36.
In this study, we engineered iMab linked to 2 copies of m36, characterized iMabm36 antiviral activity, and developed optimized variants of this novel bispecific Ab to further enhance its antiviral activity. iMabm36 has increased antiviral activity over iMab and m36 alone. We also sought to understand its dual mechanism of action because we found that the improved activity of iMabm36 was dependent on both CD4 binding and sensitivity to m36. The interdependency of this dual mechanism of action enables the high potency and breadth of iMabm36. Moreover, we investigated the influence of linker length and m36 composition and specificity on its antiviral activity for further optimizing antiviral potency and breadth.
We attribute the improved antiviral activity of iMabm36 to its interdependent, dual mechanism of action. iMabm36 binds with high affinity to CD4 via its iMab component, preconcentrating m36 on the target cell surface in the vicinity of viral entry. Engagement of HIV-1 Env with CD433,34,41 and subsequent formation and exposure of the bridging sheet of gp120 (unpublished data), both of which are unimpaired by iMab, leads to the formation of the m36 epitope, which can then be efficiently targeted by the m36 domains fused to the C-terminus of the iMab heavy chain due to the reduced steric constraints imposed by the virological synapse and thus interfere with CoR engagement. Because almost all HIV-1 isolates use CD4 as a primary entry receptor in vivo and the m36 targeting site is relatively conserved across all HIV-1 isolates, simultaneously targeting of these 2 sites provides potent antiviral activity and likely a high barrier against viral resistance. Interestingly, no antiviral activity enhancement was observed when m36 was fused to the N-terminal of the heavy or light chain of iMab (data not shown). On the other hand, fusion of m36 to the C-terminal of iMab may block the adjacent “entry complex” of gp120 and CoR interaction. As such, the unique location and enriched local concentration of m36 when fused to the C-terminal of iMab may overcome temporal and steric restrictions during viral entry, thus further enhancing antiviral potency and breadth as compared with either m36 or iMab alone.
Although the CD4i Abs contact a relatively conserved gp120 element, changes in the major gp120 variable loops can influence the activity of these CD4i Abs.27,42 Our studies showed that viruses with clade A and clade G Envs were resistant to iMabm36 neutralization. However, mutating the bridging sheet of some Clade A viruses (Q23.17, Q259.d2.17, and T28-50) to resemble that of clade B viruses can render them sensitive to iMabm36. Such neutralization differences between these viruses and their mutants provide a natural explanation for the limited sensitivity of clade A viruses to neutralization by iMabm36. m36 was selected based on clade B Envs (JRFL, Bal, and R2),26,43 and our results indicate that the activity of iMabm36 is partially determined by the virus sensitivity to m36. Therefore, further modification of m36, that is, selection based on divergent Envs, might be a potential way to further enhance the antiviral activity of the iMabm36 bibNAb.
Previous studies showed that the limited patches of conserved sequence on the CoR-binding surface are available to be accessed by CD4i antibodies.25 The linker length between m36 and iMab may restrict the flexibility of m36 to access its target. Indeed, the results obtained using constructs with different linker lengths indicate that lengthening the linker between iMab and m36 could improve the MPI against the resistant viruses. One possibility could be that the longer linker provides m36 with greater flexibility to adequately cover and recognize the binding site more efficiently than that of a short linker.
It is reported that CD4i antibodies include 2 groups based on antibody CDR length.21,35 In the CDR short group (CDR H3 10–14 AA), the CDR H3s were not very acidic, whereas the CDR H2s displayed an unusual concentration of 3 or 4 acidic residues at the loop tip. In the CDR long group (CDR H3 19–25 AA), the CDR H3s were acidic and comprised more tyrosines in contrast to those in the short group. Because the conserved CD4i epitope component is located at the interface of the gp120 outer domain and bridging sheet, electrostatic interactions provided by net charge could influence its interaction with gp120 and drive conformational changes related to virus entry. Thus, through the CDR H3 (E51) substitution,38,44,45 an improved version of iMabm36(CDR3 E51) was generated that achieves a greater MPI. These data suggest CDR H3 modifications similar to those of the tyrosine-rich and acidic N-terminal region of CCR5 may provide better recognition and binding to gp120 than in unmodified iMabm36. This approach indicates that such an iMabm36 CDRH optimization, even though slightly, might provide tyrosine posttranslational mimicry of the CCR5 N-terminus and enhanced electrostatic interaction to reduce the binding energy between the modified m36 CDR and gp120, especially when the high local concentration of m36 was achieved by anchoring it near the CoR-binding site by iMab or PRO140.
Strategies to combine bNAbs against HIV-1 have been explored to confront the emergence of resistant mutants, and bispecific antibodies continue to be an area of great interest in the pursuit of next-generation monoclonal antibodies against disease. Rationally designed, dual-targeting fusion antibodies, such as our previously described PG9-iMab, PG16-iMab, and now iMabm36 and PRO140m36, may improve the antiviral activity of bNAbs by anchoring bNAb activity at the site of viral entry, enhancing both their local concentration and accessibility to their cognate epitope. Such bibNAbs may improve the overall activity of bNAbs and antagonize HIV-1 escape better than antibody combinations and could provide a high barrier against HIV-1 escape.
The authors would like to thank Francine McCutchan, Beatrice Hahn, David Montefiori, Michael Thomson, Ronald Swanstrom, Lynn Morris, Jerome Kim, Linqi Zhang, Dennis Ellenberger, and Carolyn Williamson for contributing HIV-1 envelope plasmids used in the CAVD virus panel, and Elise Zablowsky for performing neutralization assays.
1. Fast track for antiretroviral. AIDS Patient Care and STDs. 2003;17:665.
2. Wu X, Yang ZY, Li Y, et al.. Rational design of envelope identifies broadly neutralizing human monoclonal antibodies to HIV-1. Science. 2010;329:856–861.
3. Diskin R, Scheid JF, Marcovecchio PM, et al.. Increasing the potency and breadth of an HIV antibody by using structure-based rational design. Science. 2011;334:1289–1293.
4. Pejchal R, Doores KJ, Walker LM, et al.. A potent and broad neutralizing antibody recognizes and penetrates the HIV glycan shield. Science. 2011;334:1097–1103.
5. Huang J, Ofek G, Laub L, et al.. Broad and potent neutralization of HIV-1 by a gp41-specific human antibody. Nature. 2012;491:406–412.
6. Walker LM, Phogat SK, Chan-Hui PY, et al.. Broad and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target. Science. 2009;326:285–289.
7. Moldt B, Rakasz EG, Schultz N, et al.. Highly potent HIV-specific antibody neutralization in vitro translates into effective protection against mucosal SHIV challenge in vivo. Proc Natl Acad Sci U S A. 2012;109:18921–18925.
8. Barouch DH, Whitney JB, Moldt B, et al.. Therapeutic efficacy of potent neutralizing HIV-1-specific monoclonal antibodies in SHIV-infected rhesus monkeys. Nature. 2013;503:224–228.
9. Chen W, Dimitrov DS. Human monoclonal antibodies and engineered antibody domains as HIV-1 entry inhibitors. Curr Opin HIV AIDS. 2009;4:112–117.
10. Klein F, Halper-Stromberg A, Horwitz JA, et al.. HIV therapy by a combination of broadly neutralizing antibodies in humanized mice. Nature. 2012;492:118–122.
11. Shingai M, Nishimura Y, Klein F, et al.. Antibody-mediated immunotherapy of macaques chronically infected with SHIV suppresses viraemia. Nature. 2013;503:277–280.
12. Chen W, Dimitrov DS. Monoclonal antibody-based candidate therapeutics against HIV type 1. AIDS Res Hum Retroviruses. 2012;28:425–434.
13. Armbruster C, Stiegler GM, Vcelar BA, et al.. A phase I trial with two human monoclonal antibodies (hMAb 2F5, 2G12) against HIV-1. AIDS. 2002;16:227–233.
14. Ferrantelli F, Hofmann-Lehmann R, Rasmussen RA, et al.. Post-exposure prophylaxis with human monoclonal antibodies prevented SHIV89.6P infection or disease in neonatal macaques. AIDS. 2003;17:301–309.
15. Horwitz JA, Halper-Stromberg A, Mouquet H, et al.. HIV-1 suppression and durable control by combining single broadly neutralizing antibodies and antiretroviral drugs in humanized mice. Proc Natl Acad Sci U S A. 2013;110:16538–16543.
16. Kwong PD, Wyatt R, Robinson J, et al.. Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature. 1998;393:648–659.
17. Dalgleish AG, Beverley PC, Clapham PR, et al.. The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus. Nature. 1985;312:763–767.
18. Moore JP, Doms RW. The entry of entry inhibitors: a fusion of science and medicine. Proc Natl Acad Sci U S A. 2003;100:10598–10602.
19. Liu J, Bartesaghi A, Borgnia MJ, et al.. Molecular architecture of native HIV-1 gp120 trimers. Nature. 2008;455:109–113.
20. Harris A, Borgnia MJ, Shi D, et al.. Trimeric HIV-1 glycoprotein gp140 immunogens and native HIV-1 envelope glycoproteins display the same closed and open quaternary molecular architectures. Proc Natl Acad Sci U S A. 2011;108:11440–11445.
21. Huang CC, Venturi M, Majeed S, et al.. Structural basis of tyrosine sulfation and VH-gene usage in antibodies that recognize the HIV type 1 coreceptor-binding site on gp120. Proc Natl Acad Sci U S A. 2004;101:2706–2711.
22. Meyerson JR, Tran EE, Kuybeda O, et al.. Molecular structures of trimeric HIV-1 Env in complex with small antibody derivatives. Proc Natl Acad Sci U S A. 2013;110:513–518.
23. Walker LM, Huber M, Doores KJ, et al.. Broad neutralization coverage of HIV by multiple highly potent antibodies. Nature. 2011;477:466–470.
24. Forsman A, Beirnaert E, Aasa-Chapman MM, et al.. Llama antibody fragments with cross-subtype human immunodeficiency virus type 1 (HIV-1)-neutralizing properties and high affinity for HIV-1 gp120. J Virol. 2008;82:12069–12081.
25. Labrijn AF, Poignard P, Raja A, et al.. Access of antibody molecules to the conserved coreceptor binding site on glycoprotein gp120 is sterically restricted on primary human immunodeficiency virus type 1. J Virol. 2003;77:10557–10565.
26. Chen W, Zhu Z, Feng Y, et al.. Human domain antibodies to conserved sterically restricted regions on gp120 as exceptionally potent cross-reactive HIV-1 neutralizers. Proc Natl Acad Sci U S A. 2008;105:17121–17126.
27. Wan C, Sun J, Chen W, et al.. Epitope mapping of M36, a human antibody domain with potent and broad HIV-1 inhibitory activity. PloS One. 2013;8:e66638.
28. Chen W, Xiao X, Wang Y, et al.. Bifunctional fusion proteins of the human engineered antibody domain m36 with human soluble CD4 are potent inhibitors of diverse HIV-1 isolates. Antivir Res. 2010;88:107–115.
29. Jacobson JM, Kuritzkes DR, Godofsky E, et al.. Safety, pharmacokinetics, and antiretroviral activity of multiple doses of ibalizumab (formerly TNX-355), an anti-CD4 monoclonal antibody, in human immunodeficiency virus type 1-infected adults. Antimicrob Agents Chemother. 2009;53:450–457.
30. Kuritzkes DR, Jacobson J, Powderly WG, et al.. Antiretroviral activity of the anti-CD4 monoclonal antibody TNX-355 in patients infected with HIV type 1. J Infect Dis. 2004;189:286–291.
31. Pace CS, Fordyce MW, Franco D, et al.. Anti-CD4 monoclonal antibody ibalizumab exhibits breadth and potency against HIV-1, with natural resistance mediated by the loss of a V5 glycan in envelope. J Acquir Immune Defic Syndr. 2013;62:1–9.
32. Burkly LC, Olson D, Shapiro R, et al.. Inhibition of HIV infection by a novel CD4 domain 2-specific monoclonal antibody. Dissecting the basis for its inhibitory effect on HIV-induced cell fusion. J Immunol. 1992;149:1779–1787.
33. Song R, Franco D, Kao CY, et al.. Epitope mapping of ibalizumab, a humanized anti-CD4 monoclonal antibody with anti-HIV-1 activity in infected patients. J Virol. 2010;84:6935–6942.
34. Freeman MM, Seaman MS, Rits-Volloch S, et al.. Crystal structure of HIV-1 primary receptor CD4 in complex with a potent antiviral antibody. Structure. 2010;18:1632–1641.
35. Choe H, Li W, Wright PL, et al.. Tyrosine sulfation of human antibodies contributes to recognition of the CCR5 binding region of HIV-1 gp120. Cell. 2003;114:161–170.
36. Seaman MS, Janes H, Hawkins N, et al.. Tiered categorization of a diverse panel of HIV-1 Env pseudoviruses for assessment of neutralizing antibodies. J Virol. 2010;84:1439–1452.
37. Trkola A, Ketas TJ, Nagashima KA, et al.. Potent, broad-spectrum inhibition of human immunodeficiency virus type 1 by the CCR5 monoclonal antibody PRO 140. J Virol. 2001;75:579–588.
38. West AP Jr, Galimidi RP, Foglesong CP, et al.. Evaluation of CD4-CD4i antibody architectures yields potent, broadly cross-reactive anti-human immunodeficiency virus reagents. J Virol. 2010;84:261–269.
39. Xiang SH, Wang L, Abreu M, et al.. Epitope mapping and characterization of a novel CD4-induced human monoclonal antibody capable of neutralizing primary HIV-1 strains. Virology. 2003;315:124–134.
40. Weinberg J, Liao HX, Torres JV, et al.. Identification of a synthetic peptide that mimics an HIV glycoprotein 120 envelope conformational determinant exposed following ligation of glycoprotein 120 by CD4. AIDS Res Hum Retroviruses. 1997;13:657–664.
41. Moore JP, Sattentau QJ, Klasse PJ, et al.. A monoclonal antibody to CD4 domain 2 blocks soluble CD4-induced conformational changes in the envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) and HIV-1 infection of CD4+ cells. J Virol. 1992;66:4784–4793.
42. Xiang SH, Doka N, Choudhary RK, et al.. Characterization of CD4-induced epitopes on the HIV type 1 gp120 envelope glycoprotein recognized by neutralizing human monoclonal antibodies. AIDS Res Hum Retroviruses. 2002;18:1207–1217.
43. Chen L, Kwon YD, Zhou T, et al.. Structural basis of immune evasion at the site of CD4 attachment on HIV-1 gp120. Science. 2009;326:1123–1127.
44. Dorfman T, Moore MJ, Guth AC, et al.. A tyrosine-sulfated peptide derived from the heavy-chain CDR3 region of an HIV-1-neutralizing antibody binds gp120 and inhibits HIV-1 infection. J Biol Chem. 2006;281:28529–28535.
45. Kwong PD, Wyatt R, Majeed S, et al.. Structures of HIV-1 gp120 envelope glycoproteins from laboratory-adapted and primary isolates. Structure. 2000;8:1329–1339.
HIV; neutralizing antibodies; bispecific antibody; antiretroviral; ibalizumab (iMab); m36; entry inhibitors
© 2014 by Lippincott Williams & Wilkins
What does "Remember me" mean?
By checking this box, you'll stay logged in until you logout. You'll get easier access to your articles, collections,
media, and all your other content, even if you close your browser or shut down your
To protect your most sensitive data and activities (like changing your password),
we'll ask you to re-enter your password when you access these services.
What if I'm on a computer that I share with others?
If you're using a public computer or you share this computer with others, we recommend
that you uncheck the "Remember me" box.
Highlight selected keywords in the article text.
Data is temporarily unavailable. Please try again soon.
Readers Of this Article Also Read