Anti-HIV Double Variable Domain Immunoglobulins Binding Both gp41 and gp120 for Targeted Delivery of Immunoconjugates

Craig, Ryan B.; Summa, Christopher M.; Corti, Miriam; Pincus, Seth H.

doi:10.1371/journal.pone.0046778

Cited by 24 publications

(42 citation statements)

References 68 publications

(83 reference statements)

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“…To facilitate the screening of MAbs for their efficacy as ITs, we used an indirect assay in which cells were first incubated with a given MAb and then incubated with anti-human IgG conjugated to ricin A chain (RAC). This assay has been shown to be highly predictive in determining whether a MAb will make an effective IT when it is conjugated to RAC (36)(37)(38) (Fig. 1A).…”

Section: Resultsmentioning

confidence: 99%

“…An indirect immunotoxin cytotoxicity assay was performed to screen unconjugated antibodies to predict their potential ability to kill infected cells (36)(37)(38). Uninfected H9 (1.5 ϫ 10 4 ), H9/NL4-3 (8 ϫ10 3 ), 293T (3 ϫ 10 4 ), 293T/92UG (3 ϫ 10 4 ), or 293T/C97 (3 ϫ 10 4 ) cells were plated in triplicate in RPMI 1640 medium or Dulbecco modified Eagle medium plus 10% fetal bovine serum in 96-well flat-bottom tissue culture plates (Costar).…”

Section: Methodsmentioning

confidence: 99%

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Identification of Human Anti-HIV gp160 Monoclonal Antibodies That Make Effective Immunotoxins

Pincus

Song

Maresh

et al. 2017

J Virol

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The envelope (Env) glycoprotein of HIV is the only intact viral protein expressed on the surface of both virions and infected cells. Env is the target of neutralizing antibodies (Abs) and has been the subject of intense study in efforts to produce HIV vaccines. Therapeutic anti-Env Abs can also exert antiviral effects via Fcmediated effector mechanisms or as cytotoxic immunoconjugates, such as immunotoxins (ITs). In the course of screening monoclonal antibodies (MAbs) for their ability to deliver cytotoxic agents to infected or Env-transfected cells, we noted disparities in their functional activities. Different MAbs showed diverse functions that did not correlate with each other. For example, MAbs against the external loop region of gp41 made the most effective ITs against infected cells but did not neutralize virus and bound only moderately to the same cells that they killed so effectively when they were used in ITs. There were also differences in IT-mediated killing among transfected and infected cell lines that were unrelated to the binding of the MAb to the target cells. Our studies of a well-characterized antigen demonstrate that MAbs against different epitopes have different functional activities and that the binding of one MAb can influence the interaction of other MAbs that bind elsewhere on the antigen. These results have implications for the use of MAbs and ITs to kill HIVinfected cells and eradicate persistent reservoirs of HIV infection.IMPORTANCE There is increased interest in using antibodies to treat and cure HIV infection. Antibodies can neutralize free virus and kill cells already carrying the virus. The virus envelope (Env) is the only HIV protein expressed on the surfaces of virions and infected cells. In this study, we examined a panel of human anti-Env antibodies for their ability to deliver cell-killing toxins to HIV-infected cells and to perform other antiviral functions. The ability of an antibody to make an effective immunotoxin could not be predicted from its other functional characteristics, such as its neutralizing activity. Anti-HIV immunotoxins could be used to eliminate virus reservoirs that persist despite effective antiretroviral therapy.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Identification of Human Anti-HIV gp160 Monoclonal Antibodies That Make Effective Immunotoxins

Pincus

Song

Maresh

et al. 2017

J Virol

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“…Both structures were determined by molecular replacement using the GRASP55 structure (Protein Data Bank code 3RLE (17)) as a search model. The preliminary models were refined using the Phenix program (32), and manual adjustments of residue positions were performed in Coot (33). The data collection and refinement statistics are summarized in Table 1.…”

Section: Methodsmentioning

confidence: 99%

Structural Insight into Golgi Membrane Stacking by GRASP65 and GRASP55 Proteins

Feng

et al. 2013

Journal of Biological Chemistry

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Background:The oligomerization of GRASP65 and GRASP55 is required to tether Golgi membranes. Results: The crystal structures reveal two types of intermolecular interactions, and biochemical and cellular assays confirm these observations. Conclusion: Two relatively weak interactions in combination are needed for GRASP-mediated Golgi stacking. Significance: These data suggest a novel mode of Golgi membrane stacking by the GRASP proteins.

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“…Anti-UCHL1 antibody was detected in macaque plasma by ELISA [24]. Briefly, ELISA plates were coated with 1µg/ml UCHL1 overnight at 4 ° C before blocking in blotto buffer.…”

Section: Methodsmentioning

confidence: 99%

Expression of the Memory Marker CD45RO on Helper T Cells in Macaques

et al. 2013

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BackgroundIn humans it has been reported that a major site of the latent reservoir of HIV is within CD4+ T cells expressing the memory marker CD45RO, defined by the mAb UCHL1. There are conflicting reports regarding the expression of this antigen in macaques, the most relevant animal species for studying HIV pathogenesis and testing new therapies. There is now a major effort to eradicate HIV reservoirs and cure the infection. One approach is to eliminate subsets of cells housing the latent reservoir, using UCHL1 to target these cells. So that such studies may be performed in macaques, it is essential to determine expression of CD45RO.MethodsWe have used immunofluorescence and flow cytometry to study cell surface expression of CD45RO on lymphocytes from PBMC, lymphoid, and GI organs of rhesus, pigtailed, and cynomolgus macaques. Both direct and indirect immunofluorescence experiments were performed.FindingsCD45RO is expressed on a subset of CD4+ lymphocytes of all pigtailed, a fraction of rhesus, and neither of the cynomolgus macaques studied. The binding of UCHL1 to macaque cells was of lower avidity than to human cells. This could be overcome by forming UCHL1 multimers. Directly conjugating fluors to UCHL1 can inhibit UCHL1 binding to macaque cells. Patterns of UCHL1 expression differ somewhat in macaques and humans, and from that of other memory markers often used in macaques.ConclusionsCD45RO, defined with mAb UCHL1, is well expressed on CD4+ cells in pigtailed macaques. Using tissues recovered from latently infected pigtailed macaques we are determining whether UCHL1, or other memory markers, can define the cellular locus of the reservoir. The low avidity of this interaction could limit the utility of UCHL1, in its conventional form, to eliminate cells in vivo and test this approach in macaque models of HIV infection.

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Anti-HIV Double Variable Domain Immunoglobulins Binding Both gp41 and gp120 for Targeted Delivery of Immunoconjugates

Cited by 24 publications

References 68 publications

Identification of Human Anti-HIV gp160 Monoclonal Antibodies That Make Effective Immunotoxins

Identification of Human Anti-HIV gp160 Monoclonal Antibodies That Make Effective Immunotoxins

Structural Insight into Golgi Membrane Stacking by GRASP65 and GRASP55 Proteins

Expression of the Memory Marker CD45RO on Helper T Cells in Macaques

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