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1.  An Anti-PCSK9 Antibody Reduces LDL-Cholesterol On Top Of A Statin And Suppresses Hepatocyte SREBP-Regulated Genes 
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a promising therapeutic target for treating coronary heart disease. We report a novel antibody 1B20 that binds to PCSK9 with sub-nanomolar affinity and antagonizes PCSK9 function in-vitro. In CETP/LDLR-hemi mice two successive doses of 1B20, administered 14 days apart at 3 or 10 mpk, induced dose dependent reductions in LDL-cholesterol (≥ 25% for 7-14 days) that correlated well with the extent of PCSK9 occupancy by the antibody. In addition, 1B20 induces increases in total plasma antibody-bound PCSK9 levels and decreases in liver mRNA levels of SREBP-regulated genes PCSK9 and LDLR, with a time course that parallels decreases in plasma LDL-cholesterol (LDL-C). Consistent with this observation in mice, in statin-responsive human primary hepatocytes, 1B20 lowers PCSK9 and LDLR mRNA levels and raises serum steady-state levels of antibody-bound PCSK9. In addition, mRNA levels of several SREBP regulated genes involved in cholesterol and fatty-acid synthesis including ACSS2, FDPS, IDI1, MVD, HMGCR, and CYP51A1 were decreased significantly with antibody treatment of primary human hepatocytes. In rhesus monkeys, subcutaneous (SC) dosing of 1B20 dose-dependently induces robust LDL-C lowering (maximal ~70%), which is correlated with increases in target engagement and total antibody-bound PCSK9 levels. Importantly, a combination of 1B20 and Simvastatin in dyslipidemic rhesus monkeys reduced LDL-C more than either agent alone, consistent with a mechanism of action that predicts additive effects of anti-PCSK9 agents with statins. Our results suggest that antibodies targeting PCSK9 could provide patients powerful LDL lowering efficacy on top of statins, and lower cardiovascular risk.
doi:10.7150/ijbs.3524
PMCID: PMC3282994  PMID: 22355267
PCSK9; low density lipoprotein cholesterol (LDL-C); hypercholesterolemia; LDL receptor; sterol regulatory element binding protein (SREBP); primary hepatocytes
2.  IgG2m4, an engineered antibody isotype with reduced Fc function 
mAbs  2009;1(6):572-579.
The Fc region of an antibody mediates effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and plays a key role in the in vivo half-life of an antibody. In designing antibody therapeutics, it is sometimes desirable that the antibody has altered Fc-mediated properties. In the case of a “benign blocker” antibody, it is often desirable to diminish or abolish the ADCC and CDC functions while retaining its PK profile. Here, we report a novel engineered IgG isotype, IgG2m4, with reduced Fc functionality. IgG2m4 is based on the IgG2 isotype with four key amino acid residue changes derived from IgG4 (H268Q, V309L, A330S and P331S). An IgG2m4 antibody has an overall reduction in complement and Fcγ receptor binding in in vitro binding analyses while maintaining the normal in vivo serum half-life in rhesus.
PMCID: PMC2791314  PMID: 20073128
IgG2m4; IgG; isotype; benign blocker; Fcγ receptors; C1q
3.  Affinity maturation and characterization of a human monoclonal antibody against HIV-1 gp41 
mAbs  2009;1(5):462-474.
The human D5 monoclonal antibody binds to the highly conserved hydrophobic pocket on the N-terminal heptad repeat (NHR) trimer of HIV-1 gp41 and exhibits modest yet relatively broad neutralization activity. Both binding and neutralization depend on residues in the complementarity determining regions (CDRs) of the D5 IgG variable domains on heavy chain (VH) and light chain (VL). In an effort to increase neutralization activity to a wider range of HIV-1 strains, we have affinity matured the parental D5 scFv by randomizing selected residues in 5 of its 6 CDRs. The resulting scFv variants derived from four different CDR changes showed enhanced binding affinities to gp41 NHR mimetic (5-helix) which correlated to improved neutralization potencies by up to 8-fold. However, when converted to IgG1s, these D5 variants had up to a 12-fold reduction in neutralization potency over their corresponding scFvs despite their slightly enhanced in vitro binding affinities. Remarkably, D5 variant IgG1s bearing residue changes in CDRs that interact with epitope residues N-terminal to the hydrophobic pocket (such as VH CDR3 and VL CDR3) retained more neutralization potency than those containing residue changes in pocket-interacting CDRs (such as VH CDR2). These results provide compelling evidence for the existence of a steric block to an IgG that extends to the gp41 NHR hydrophobic pocket region, and can be a useful guide for developing therapeutic antibodies and vaccines circumventing this block.
PMCID: PMC2759496  PMID: 20065653
affinity maturation; monoclonal antibody; scFv; gp41; HIV-1; neutralization
4.  Efficacy of Multivalent Adenovirus-Based Vaccine against Simian Immunodeficiency Virus Challenge ▿  
Journal of Virology  2009;84(6):2996-3003.
The prophylactic efficacies of several multivalent replication-incompetent adenovirus serotype 5 (Ad5) vaccines were examined in rhesus macaques using an intrarectal high-dose simian immunodeficiency virus SIVmac239 challenge model. Cohorts of Mamu-A*01+/B*17− Indian rhesus macaques were immunized with one of several combinations of Ad5 vectors expressing Gag, Pol, Nef, and Env gp140; for comparison, a Mamu-A*01+ cohort was immunized using the Ad5 vector alone. There was no sign of immunological interference between antigens in the immunized animals. In general, expansion of the antigen breadth resulted in more favorable virological outcomes. In particular, the order of efficacy trended as follows: Gag/Pol/Nef/Env ≈ Gag/Pol > Gag ≈ Gag/Pol/Nef > Nef. However, the precision in ranking the vaccines based on the study results may be limited by the cohort size, and as such, may warrant additional testing. The implications of these results in light of the recent discouraging results of the phase IIb study of the trivalent Ad5 HIV-1 vaccine are discussed.
doi:10.1128/JVI.00969-09
PMCID: PMC2826028  PMID: 20042509
6.  Distinct expression profiles of Notch-1 protein in human solid tumors: Implications for development of targeted therapeutic monoclonal antibodies 
Biological therapies, such as monoclonal antibodies (mAbs) that target tumor-associated antigens have been considered an effective therapeutic approach in oncology. In considering Notch-1 receptor as a potential target, we performed immunohistochemistry on tissue microarrays to determine 1) whether the receptor is overexpressed in tumor cells as compared to their corresponding normal tissues and 2) the clinical significance of its expression levels in human breast, colorectal, lung and prostate cancers. We found that the expression of Notch-1 protein was overexpressed in primary colorectal adenocarcinoma and nonsmall cell lung carcinoma (NSCLC), but not in primary ductal breast carcinoma or prostate adenocarcinoma. Further analysis revealed that higher levels of Notch-1 protein expression were significantly associated with poorer differentiation of breast and prostate tumors. Strikingly, for NSCLC, the expression levels of Notch-1 protein were found to be inversely correlated with tumor differentiation and progression. For colorectal tumors, however, no correlation of Notch-1 protein expression was found with any tumor clinicopathological parameters, in spite of its overexpression in tumor cells. Our data demonstrated the complexity of Notch-1 protein expression in human solid tumors and further supported the notion that the roles of Notch-1 expression in tumorigenesis are highly context-dependent. The findings could provide the basis for development of distinct therapeutic strategies of Notch-1 mAbs for its applications in the treatment of suitable types of human cancers.
PMCID: PMC2898104  PMID: 20631820
Notch-1; target therapy; tissue microarray; immunohistochemistry
7.  Safety and Immunogenicity of the Merck Adenovirus Serotype 5 (MRKAd5) and MRKAd6 Human Immunodeficiency Virus Type 1 Trigene Vaccines Alone and in Combination in Healthy Adults▿  
Preexisting immunity to adenovirus serotype 5 (Ad5) diminishes immune responses to vaccines using Ad5 as a vector. Alternate Ad serotypes as vaccine vectors might overcome Ad5-specific neutralizing antibodies and enhance immune responses in populations with a high prevalence of Ad5 immunity. To test this hypothesis, healthy human immunodeficiency virus (HIV)-seronegative adults were enrolled in a blinded, randomized, dose-escalating, placebo-controlled study. In part A, subjects with baseline Ad6 titers of ≤18 received the Merck Ad6 (MRKAd6) HIV type 1 (HIV-1) trigene vaccine at weeks 0, 4, and 26. In part B, subjects stratified by Ad5 titers (≤200 or >200) and Ad6 titers (≤18 or >18) received the MRKAd5-plus-MRKAd6 (MRKAd5+6) HIV-1 trigene vaccine at weeks 0, 4, and 26. Immunogenicity was assessed by an enzyme-linked immunospot (ELISPOT) assay at week 30. No serious adverse events occurred. MRKAd6 trigene vaccine recipients responded more often to Nef than to Gag or Pol. In part A, ELISPOT response rates to ≥2 vaccine antigens were 14%, 63%, and 71% at 109, 1010, and 1011 viral genomes (vg)/dose, respectively. All responders had positive Nef-specific ELISPOT results. In part B, Nef-ELISPOT response rates at 1010 vg/dose of the MRKAd5+6 trigene vaccine were 50% in the low-Ad5/low-Ad6 stratum (n = 8), 78% in the low-Ad5/high-Ad6 stratum (n = 9), 75% in the high-Ad5/low-Ad6 stratum (n = 8), and 44% in the high-Ad5/high-Ad6 stratum (n = 9). The MRKAd6 and MRKAd5+6 trigene vaccines elicited dose-dependent responses predominantly to Nef and were generally well tolerated, indicating that Ad6 should be considered a candidate vector for future vaccines. Although small sample sizes limit the conclusions that can be drawn from this exploratory study, combining two Ad vectors may be a useful vaccine strategy for circumventing isolated immunity to a single Ad serotype.
doi:10.1128/CVI.00144-09
PMCID: PMC2745015  PMID: 19605598
8.  Characterization of Notch1 Antibodies That Inhibit Signaling of Both Normal and Mutated Notch1 Receptors 
PLoS ONE  2010;5(2):e9094.
Background
Notch receptors normally play a key role in guiding a variety of cell fate decisions during development and differentiation of metazoan organisms. On the other hand, dysregulation of Notch1 signaling is associated with many different types of cancer as well as tumor angiogenesis, making Notch1 a potential therapeutic target.
Principal Findings
Here we report the in vitro activities of inhibitory Notch1 monoclonal antibodies derived from cell-based and solid-phase screening of a phage display library. Two classes of antibodies were found, one directed against the EGF-repeat region that encompasses the ligand-binding domain (LBD), and the second directed against the activation switch of the receptor, the Notch negative regulatory region (NRR). The antibodies are selective for Notch1, inhibiting Jag2-dependent signaling by Notch1 but not by Notch 2 and 3 in reporter gene assays, with EC50 values as low as 5±3 nM and 0.13±0.09 nM for the LBD and NRR antibodies, respectively, and fail to recognize Notch4. While more potent, NRR antibodies are incomplete antagonists of Notch1 signaling. The antagonistic activity of LBD, but not NRR, antibodies is strongly dependent on the activating ligand. Both LBD and NRR antibodies bind to Notch1 on human tumor cell lines and inhibit the expression of sentinel Notch target genes, including HES1, HES5, and DTX1. NRR antibodies also strongly inhibit ligand-independent signaling in heterologous cells transiently expressing Notch1 receptors with diverse NRR “class I” point mutations, the most common type of mutation found in human T-cell acute lymphoblastic leukemia (T-ALL). In contrast, NRR antibodies failed to antagonize Notch1 receptors bearing rare “class II” or “class III” mutations, in which amino acid insertions generate a duplicated or constitutively sensitive metalloprotease cleavage site. Signaling in T-ALL cell lines bearing class I mutations is partially refractory to inhibitory antibodies as compared to cell-penetrating gamma-secretase inhibitors.
Conclusions/Significance
Antibodies that compete with Notch1 ligand binding or that bind to the negative regulatory region can act as potent inhibitors of Notch1 signaling. These antibodies may have clinical utility for conditions in which inhibition of signaling by wild-type Notch1 is desired, but are likely to be of limited value for treatment of T-ALLs associated with aberrant Notch1 activation.
doi:10.1371/journal.pone.0009094
PMCID: PMC2817004  PMID: 20161710
9.  Vaccine-Induced Cellular Responses Control Simian Immunodeficiency Virus Replication after Heterologous Challenge▿ †  
Journal of Virology  2009;83(13):6508-6521.
All human immunodeficiency virus (HIV) vaccine efficacy trials to date have ended in failure. Structural features of the Env glycoprotein and its enormous variability have frustrated efforts to induce broadly reactive neutralizing antibodies. To explore the extent to which vaccine-induced cellular immune responses, in the absence of neutralizing antibodies, can control replication of a heterologous, mucosal viral challenge, we vaccinated eight macaques with a DNA/Ad5 regimen expressing all of the proteins of SIVmac239 except Env. Vaccinees mounted high-frequency T-cell responses against 11 to 34 epitopes. We challenged the vaccinees and eight naïve animals with the heterologous biological isolate SIVsmE660, using a regimen intended to mimic typical HIV exposures resulting in infection. Viral loads in the vaccinees were significantly less at both the peak (1.9-log reduction; P < 0.03) and at the set point (2.6-log reduction; P < 0.006) than those in control naïve animals. Five of eight vaccinated macaques controlled acute peak viral replication to less than 80,000 viral RNA (vRNA) copy eq/ml and to less than 100 vRNA copy eq/ml in the chronic phase. Our results demonstrate that broad vaccine-induced cellular immune responses can effectively control replication of a pathogenic, heterologous AIDS virus, suggesting that T-cell-based vaccines may have greater potential than previously appreciated.
doi:10.1128/JVI.00272-09
PMCID: PMC2698536  PMID: 19403685
10.  The effect of early versus delayed challenge after vaccination in controlling SHIV 89.6P infection 
Virology  2008;381(1):75-80.
We sought to determine how effectively a CD8+ T cell inducing vaccine controls SHIV-89.6P infection in rhesus macaques at a range of challenge times post-vaccination. To this end, twenty eight Mamu-A*01+ rhesus macaques were given replication incompetent human serotype 5 adenovirus vector expressing SIVmac239 gag DNA and boosted 24 weeks later. Groups of 4 monkeys were then challenged with SHIV-89.6P at 1, 3, 6, 12, and 24 weeks after the boost. We compared the kinetics of viral load, CD4+ and virus-specific CD8+ T cells in these macaques. Measurements of CD8+ T cells taken before challenge show an exponential decay between 1 and 12 weeks following vaccination (p<0.0001). After week 12, no further decay was observed. Twenty of 24 vaccinated animals maintained more CD4+ T cells and kept their viral load at least one order of magnitude lower than the control animals throughout the chronic phase of the study. All 24 vaccinated animals survived the duration of the study. The viral and T cell kinetics over the first two weeks differed between the vaccinated groups, with more recent vaccination improving the early control of virus (p-value = 0.027). The rates of virus specific CD8+ T cell expansion were greater in animals having higher viral loads at one week (r=0.45, p = 0.029), suggesting that the kinetics of early viral load may have a role in virus specific CD8+ T cell generation, although these early differences did not lead to different clinical outcomes within the vaccinated animals.
doi:10.1016/j.virol.2008.07.042
PMCID: PMC2664825  PMID: 18793788
CD8; challenge time; vaccine; HIV; delayed response; modeling
11.  Vaccine-Induced Cellular Immune Responses Reduce Plasma Viral Concentrations after Repeated Low-Dose Challenge with Pathogenic Simian Immunodeficiency Virus SIVmac239 
Journal of Virology  2006;80(12):5875-5885.
The goal of an AIDS vaccine regimen designed to induce cellular immune responses should be to reduce the viral set point and preserve memory CD4 lymphocytes. Here we investigated whether vaccine-induced cellular immunity in the absence of any Env-specific antibodies can control viral replication following multiple low-dose challenges with the highly pathogenic SIVmac239 isolate. Eight Mamu-A*01-positive Indian rhesus macaques were vaccinated with simian immunodeficiency virus (SIV) gag, tat, rev, and nef using a DNA prime-adenovirus boost strategy. Peak viremia (P = 0.007) and the chronic phase set point (P = 0.0192) were significantly decreased in the vaccinated cohort, out to 1 year postinfection. Loss of CD4+ memory populations was also ameliorated in vaccinated animals. Interestingly, only one of the eight vaccinees developed Env-specific neutralizing antibodies after infection. The control observed was significantly improved over that observed in animals vaccinated with SIV gag only. Vaccine-induced cellular immune responses can, therefore, exert a measure of control over replication of the AIDS virus in the complete absence of neutralizing antibody and give us hope that a vaccine designed to induce cellular immune responses might control viral replication.
doi:10.1128/JVI.00171-06
PMCID: PMC1472612  PMID: 16731926
12.  Attenuation of Simian Immunodeficiency Virus SIVmac239 Infection by Prophylactic Immunization with DNA and Recombinant Adenoviral Vaccine Vectors Expressing Gag 
Journal of Virology  2005;79(24):15547-15555.
The prophylactic efficacy of DNA and replication-incompetent adenovirus serotype 5 (Ad5) vaccine vectors expressing simian immunodeficiency virus (SIV) Gag was examined in rhesus macaques using an SIVmac239 challenge. Cohorts of either Mamu-A*01(+) or Mamu-A*01(−) macaques were immunized with a DNA prime-Ad5 boost regimen; for comparison, a third cohort consisting of Mamu-A*01(+) monkeys was immunized using the Ad5 vector alone for both prime and boost. All animals, along with unvaccinated control cohorts of Mamu-A*01(+) and Mamu-A*01(−) macaques, were challenged intrarectally with SIVmac239. Viral loads were measured in both peripheral and lymphoid compartments. Only the DNA prime-Ad5-boosted Mamu-A*01(+) cohort exhibited a notable reduction in peak plasma viral load (sevenfold) as well as in early set-point viral burdens in both plasma and lymphoid tissues (10-fold) relative to those observed in the control monkeys sharing the same Mamu-A*01 allele. The degree of control in each animal correlated with the levels of Gag-specific immunity before virus challenge. However, virus control was short-lived, and indications of viral escape were evident as early as 6 months postinfection. The implications of these results in vaccine design and clinical testing are discussed.
doi:10.1128/JVI.79.24.15547-15555.2005
PMCID: PMC1315991  PMID: 16306625
13.  Vectored Gag and Env but Not Tat Show Efficacy against Simian-Human Immunodeficiency Virus 89.6P Challenge in Mamu-A*01-Negative Rhesus Monkeys 
Journal of Virology  2005;79(19):12321-12331.
Simian-human immunodeficiency virus (SHIV) challenge studies in rhesus macaques were conducted to evaluate the efficacy of adenovirus-based vaccines in the context of different major histocompatibility complex class I genetic backgrounds and different vaccine compositions. Mamu-A*01 allele-negative rhesus monkeys were immunized with one of the following vaccine constructs: (i) replication-defective recombinant adenovirus type 5 (Ad5) expressing human immunodeficiency virus type 1 (HIV-1) Tat (Ad5/HIVTat); (ii) Ad5 vector expressing simian immunodeficiency virus (SIV) Gag (Ad5/SIVGag); (iii) Ad5 vector expressing the truncated HIV-1jrfl Env, gp140 (Ad5/gp140_jrfl); (iv) Ad5 vector expressing the SHIV-89.6P gp140 (Ad5/gp140_89.6P); or (v) the combination of Ad5/SIVGag and Ad5/gp140_jrfl. Following intravenous challenge with SHIV-89.6P, only those cohorts that received vaccines expressing Gag or Env exhibited an attenuation of the acute viremia and associated CD4-cell lymphopenia. While no prechallenge neutralizing antibody titers were detectable in either Ad5/gp140-vaccinated group, an accelerated neutralizing antibody response was observed in the Ad5/gp140_89.6P-vaccinated group upon viral challenge. The set-point viral loads in the Ad5/SIVGag- and Ad5/gp140_jrfl-vaccinated groups were associated with the overall strength of the induced cellular immune responses. To examine the contribution of Mamu-A*01 allele in vaccine efficacy against SHIV-89.6P challenge, Mamu-A*01-positive monkeys were immunized with Ad5/SIVGag. Vaccine-mediated protection was significantly more pronounced in the Mamu-A*01-positive monkeys than in Mamu-A*01-negative monkeys, suggesting the strong contributions of T-cell epitopes restricted by the Mamu-A*01 molecule. The implications of these results in the development of an HIV-1 vaccine will be discussed.
doi:10.1128/JVI.79.19.12321-12331.2005
PMCID: PMC1211517  PMID: 16160159

Results 1-13 (13)