We have continued to identify, engineer and characterize human monoclonal antibodies (hmAbs) including nanohumAbs against HIV-1, emerging and biodefense-related viruses, and cancer, and conjugate some of them with nanoparticles. We have extensively evaluated the neutralizing activity and reactivity to self antigens of our most promising anti-HIV antibodies g41-specific m43, m44, m46, m47 and m48. It has been previously found that the two best characterized gp41-specific cross-reactive neutralizing human monoclonal antibodies 4E10 and 2F5 target linear epitopes in the membrane proximal external region (MPER) and exhibit reactivity to self antigens including cardiolipin (CL) and phosphatidyliserine (PS). It has been hypothesized that such polyreactivity could be a major reason for the failure to elicit 2F5 and 4E10-like cross-reactive neutralizing hmAbs by vaccine immunogens based on the MPER. We found that in PBMCs based assays one of our antibodies, m44, neutralized most of the 21 HIV-1 primary isolates from different clades with a significantly higher average potency than that of 4E10 and Z13. In contrast to 2F5, 4E10 and Z13, m44 did not bind to any significant degree to denatured gp140 and linear peptides derived from gp41, suggesting a conformational nature of the epitope. Unlike 4E10 and 2F5, m44 did not bind to CL and PS in ELISA and Biacore assays, nor bound to any of a panel of protein and DNA autoantigens in luminex assays. This is the first gp41-specific cross-reactive hmAb that does not have detectable polyreactivity. Its novel conserved conformational epitope on gp41 could be used in the design of vaccine immunogens and as a target for therapeutics. We are also developing nanohumAbs against CD4-induced epitopes on gp120. Responding to the threat of bioterrorism and to the health crisis caused by the SARS coronavirus (SARS CoV), we have continued to identify and characterize neutralizing antibodies to the biodefense-related Hendra virus (HeV) and Nipah virus (NiV), and to the SARS CoV. Previously, we reported on the isolation of henipavirus-neutralizing recombinant hmAbs. One of these antibodies, m102, which exhibited the highest level of cross-reactivity and binding to both NiV and HeV G protein, was affinity maturated by light chain shuffling, and further panning against a soluble form of the HeV G protein, sGHeV. The most matured clone, designated m102.4, was converted to IgG1 and tested against infectious NiV and HeV; it exhibited exceptionally potent and cross-reactive inhibitory activity with 50% inhibitory concentrations below 0.04 and 0.6 g/ml, respectively. This is the first and only hmAb that is exceptionally potent against both HeV and NiV. It has long (weeks) half-life and no toxicities in ferrets, and is being tested in animal models based on ferrets and cats. These results suggest that m102.4 has potential as a therapeutic for treatment of diseases caused by henipaviruses. It could be also used for prophylaxis, diagnosis and as a research reagent. The SARS CoV caused a worldwide epidemic in late 2002/early 2003 and a second outbreak in the winter of 2003-2004 by an independent animal to human transmission. The GD03 strain, which was isolated from an index patient of the second outbreak, was reported to resist neutralization by the hmAbs 80R and S3.1 which can potently neutralize isolates from the first outbreak. We have recently reported that two hmAbs, m396 and S230.15, potently neutralized GD03 and representative isolates from the first SARS outbreak (Urbani, Tor2) and from palm civets (SZ3, SZ16). These antibodies also protected mice challenged with the Urbani, or recombinant viruses bearing the GD03 and SZ16 S glycoproteins. Both antibodies competed with the SARS CoV receptor, ACE2, for binding to the RBD suggesting a mechanism of neutralization that involves interference with the SARS CoV-ACE2 interaction. Based on the previously solved RBD-m396 structure we performed extensive site directed mutagenesis of the RBD. Two putative hot spot residues in the RBD (I489 and Y491) were identified within the SARS CoV spike that likely contribute to most of the m396 binding energy. Residues I489 and Y491 are highly conserved within the SARS CoV spike indicating a possible mechanism of the m396 cross-reactivity. Sequence analysis and mutagenesis data show that m396 might neutralize all zoonotic and epidemic SARS CoV isolates with known sequences, except strains derived from bats. These antibodies are the first identified hmAbs, which exhibit cross-reactivity against isolates from the two SARS outbreaks and palm civets, and could have potential applications for diagnosis, prophylaxis and treatment of SARS CoV infections. We have also continued to characterize our antibodies against components of the insulin-like growth factor (IGF) system which plays an important role in cancer. Currently several monoclonal antibodies targeting the IGF receptor type I (IGF-IR) are being tested in patients with solid tumors. However, penetration of full-length antibodies into solid tumors is slow and inefficient. We have been hypothesizing that targeting the IGF-IR ligands, IGF-I and IGF-II, by antibodies may not require antibody presence in the tumor interstitial space but could shift the complex equilibrium and quasi-steady states to ligand-antibody complexes in the plasma leading to depletion of the ligand in the tumor. To test this hypothesis we used a fully human monoclonal antibody, IgG1 m610, which binds with high (nM) affinity to IGF-II and inhibits signal transduction mediated by the IGF-II interaction with the IGF-IR. We chose a mouse xenograft model based on MCF-7 cells because these breast cancer cells produce high levels of IGF-II but do not require this growth factor for their proliferation. The IGF-II concentration in tumors was significantly decreased from 24 +/-14 nM (range 11 - 37 nM) to 8 +/-3 nM (range 4-10 nM) after the fourth administration of the antibody at the highest concentration of 1 mg per mouse compared to the control without antibody. The decrease was dependent on the antibody concentration at 0.1 mg per mouse it was on average 1.4-fold. The IGF-II depletion in the interstitial fluid is likely to be much higher - an estimate based on 30% interstitial fluid fraction and uniform IGF-II concentration in the tumor indicates at least 10-fold decrease. The depletion was specific for IGF-II since the IGF-I concentration in the tumor was not affected. These results support the concept that full-length antibodies can decrease the concentration of soluble ligands in tumors. Because limited access to interstitial fluid in solid tumors is a major problem for the efficacy of intact antibodies, targeting ligands of growth factors could be an useful strategy for treatment of solid tumors. Targeting of IGF-II could inhibit not only its binding to the IGF-IR but also its binding to the insulin receptor and decrease cell proliferation caused by signals initiated by the IGF-II interaction with both receptors. Thus m610 could be used in combination with anti-IGF-IR antibodies and other anti-cancer drugs for treatment of IGF-II-sensitive solid tumors. We are also developing nanohumAbs against the IGF-IR and conjugating some of our antibodies with nanoliposomes. We plan to continue to identify novel potent nhmAbs against cancer, HIV-1, and biodef [summary truncated at 7800 characters]

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National Cancer Institute (NCI)
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Chen, Weizao; Xiao, Xiaodong; Wang, Yanping et al. (2010) Bifunctional fusion proteins of the human engineered antibody domain m36 with human soluble CD4 are potent inhibitors of diverse HIV-1 isolates. Antiviral Res 88:107-15
Chen, Weizao; Dimitrov, Dimiter S (2009) Human monoclonal antibodies and engineered antibody domains as HIV-1 entry inhibitors. Curr Opin HIV AIDS 4:112-7
Chen, Weizao; Zhu, Zhongyu; Feng, Yang et al. (2008) Construction of a large phage-displayed human antibody domain library with a scaffold based on a newly identified highly soluble, stable heavy chain variable domain. J Mol Biol 382:779-89
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