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Targeting HIV Reservoir in Infected CD4 T Cells by Dual-Affinity Re-targeting Molecules (DARTs) that Bind HIV Envelope and Recruit Cytotoxic T Cells.

Sloan DD, Lam CY, Irrinki A, Liu L, Tsai A, Pace CS, Kaur J, Murry JP, Balakrishnan M, Moore PA, Johnson S, Nordstrom JL, Cihlar T, Koenig S - PLoS Pathog. (2015)

Bottom Line: In an ex vivo model using cells isolated from HIV-infected participants on cART, combinations of the most potent HIVxCD3 DARTs reduced HIV expression both in quiescent and activated peripheral blood mononuclear cell cultures isolated from HIV-infected participants on suppressive cART.Importantly, HIVxCD3 DARTs did not induce cell-to-cell virus spread in resting or activated CD4 T cell cultures.Collectively, these results provide support for further development of HIVxCD3 DARTs as a promising therapeutic strategy for targeting HIV reservoirs.

View Article: PubMed Central - PubMed

Affiliation: Gilead Sciences, Foster City, California, United States of America.

ABSTRACT
HIV reservoirs and production of viral antigens are not eliminated in chronically infected participants treated with combination antiretroviral therapy (cART). Novel therapeutic strategies aiming at viral reservoir elimination are needed to address chronic immune dysfunction and non-AIDS morbidities that exist despite effective cART. The HIV envelope protein (Env) is emerging as a highly specific viral target for therapeutic elimination of the persistent HIV-infected reservoirs via antibody-mediated cell killing. Dual-Affinity Re-Targeting (DART) molecules exhibit a distinct mechanism of action via binding the cell surface target antigen and simultaneously engaging CD3 on cytotoxic T lymphocytes (CTLs). We designed and evaluated Env-specific DARTs (HIVxCD3 DARTs) derived from known antibodies recognizing diverse Env epitopes with or without broadly neutralizing activity. HIVxCD3 DARTs derived from PGT121, PGT145, A32, and 7B2, but not VRC01 or 10E8 antibodies, mediated potent CTL-dependent killing of quiescent primary CD4 T cells infected with diverse HIV isolates. Similar killing activity was also observed with DARTs structurally modified for in vivo half-life extension. In an ex vivo model using cells isolated from HIV-infected participants on cART, combinations of the most potent HIVxCD3 DARTs reduced HIV expression both in quiescent and activated peripheral blood mononuclear cell cultures isolated from HIV-infected participants on suppressive cART. Importantly, HIVxCD3 DARTs did not induce cell-to-cell virus spread in resting or activated CD4 T cell cultures. Collectively, these results provide support for further development of HIVxCD3 DARTs as a promising therapeutic strategy for targeting HIV reservoirs.

No MeSH data available.


Related in: MedlinePlus

HIVxCD3 DARTs retarget cytolytic CD3+ T-cells to Env-expressing HIV-infected CD4+ T-cells.(A) Mechanism of cytolysis. The CD3 arm (orange) of the bi-specific DART binds to CD3 at the surface of CD3+ T-cells and the HIV arm (blue) binds to HIV Env at the surface of HIV-infected CD4+ T-cells. Cell surface Env may be in the form of functional mature trimers or nonfunctional variant forms such as cleaved or uncleaved gp160 monomers or gp41 stumps [43]. DART-mediated engagement of target and effector cells results in activation of effector cell cytolytic responses and target cell killing. (B) Variety of Env epitopes targeted by HIVxCD3 DARTs. Locations on the mature HIV-1 Env trimer surface of epitopes recognized by the anti-Env Abs used as sources of the HIV binding arms of DARTs are shown. Broadly neutralizing Abs PGT121, PGT145, VRC01 and 10E8 target epitopes located in the V3 glycan (N332; green), V2 glycan (N160K, blue), CD4 binding site (CD4bs, orange) and gp41 MPER (cyan), respectively, that are preferentially expressed on functional Env trimers, whereas non-neutralizing Abs A32 and 7B2 target epitopes located in CD4-induced sites (CD4i conformation epitopes are not visible in the depicted pre-CD4 binding Env structure) and in the gp41 stalk (cyan), respectively, that are preferentially expressed on nonfunctional forms of Env. The depicted structure of Env trimer is derived from pdb 4NCO.
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ppat.1005233.g001: HIVxCD3 DARTs retarget cytolytic CD3+ T-cells to Env-expressing HIV-infected CD4+ T-cells.(A) Mechanism of cytolysis. The CD3 arm (orange) of the bi-specific DART binds to CD3 at the surface of CD3+ T-cells and the HIV arm (blue) binds to HIV Env at the surface of HIV-infected CD4+ T-cells. Cell surface Env may be in the form of functional mature trimers or nonfunctional variant forms such as cleaved or uncleaved gp160 monomers or gp41 stumps [43]. DART-mediated engagement of target and effector cells results in activation of effector cell cytolytic responses and target cell killing. (B) Variety of Env epitopes targeted by HIVxCD3 DARTs. Locations on the mature HIV-1 Env trimer surface of epitopes recognized by the anti-Env Abs used as sources of the HIV binding arms of DARTs are shown. Broadly neutralizing Abs PGT121, PGT145, VRC01 and 10E8 target epitopes located in the V3 glycan (N332; green), V2 glycan (N160K, blue), CD4 binding site (CD4bs, orange) and gp41 MPER (cyan), respectively, that are preferentially expressed on functional Env trimers, whereas non-neutralizing Abs A32 and 7B2 target epitopes located in CD4-induced sites (CD4i conformation epitopes are not visible in the depicted pre-CD4 binding Env structure) and in the gp41 stalk (cyan), respectively, that are preferentially expressed on nonfunctional forms of Env. The depicted structure of Env trimer is derived from pdb 4NCO.

Mentions: A series of bi-specific antibody constructs that bind simultaneously to HIV Env and human CD3 receptor were generated using the basic DART platform (Fig 1A) [29,30]. To maximize the breadth of Env recognition across multiple HIV isolates, complementarity determining regions (CDRs) from four bNAbs (PGT121, PGT145, 10E8, VRC01) [31–33] were incorporated into the Env-recognizing arm of HIVxCD3 DARTs. These bNAb-derived DARTs were compared with ones with HIV Env arms derived from two non-neutralizing antibodies (A32, 7B2) that bind broadly conserved residues in Env and efficiently induce antibody dependent cell-mediated cytotoxicity (ADCC) [34–41]. Each of the six designed bi-specific Abs recognizes a distinct epitope on the surface of HIV Env protein (Fig 1B). CDRs derived from palivizumab [42], an antibody recognizing the fusion protein of respiratory syncytial virus (RSV), were used to construct a negative control DART (RSVxCD3) that does not bind to HIV Env. The CD3-recognizing arm, which was identical in all DARTs, was derived from hXR32 [28], a humanized mouse anti-human CD3ε antibody, which cross-reacts with nonhuman primate CD3ε. The HIVxCD3 DARTs in basic format were produced by expression in stably transfected CHO cells and purified. The formation of properly assembled molecules was confirmed by reducing and non-reducing SDS-PAGE and analytical SEC; the average purity of the assembled HIVxCD3 DART molecules was 95%.


Targeting HIV Reservoir in Infected CD4 T Cells by Dual-Affinity Re-targeting Molecules (DARTs) that Bind HIV Envelope and Recruit Cytotoxic T Cells.

Sloan DD, Lam CY, Irrinki A, Liu L, Tsai A, Pace CS, Kaur J, Murry JP, Balakrishnan M, Moore PA, Johnson S, Nordstrom JL, Cihlar T, Koenig S - PLoS Pathog. (2015)

HIVxCD3 DARTs retarget cytolytic CD3+ T-cells to Env-expressing HIV-infected CD4+ T-cells.(A) Mechanism of cytolysis. The CD3 arm (orange) of the bi-specific DART binds to CD3 at the surface of CD3+ T-cells and the HIV arm (blue) binds to HIV Env at the surface of HIV-infected CD4+ T-cells. Cell surface Env may be in the form of functional mature trimers or nonfunctional variant forms such as cleaved or uncleaved gp160 monomers or gp41 stumps [43]. DART-mediated engagement of target and effector cells results in activation of effector cell cytolytic responses and target cell killing. (B) Variety of Env epitopes targeted by HIVxCD3 DARTs. Locations on the mature HIV-1 Env trimer surface of epitopes recognized by the anti-Env Abs used as sources of the HIV binding arms of DARTs are shown. Broadly neutralizing Abs PGT121, PGT145, VRC01 and 10E8 target epitopes located in the V3 glycan (N332; green), V2 glycan (N160K, blue), CD4 binding site (CD4bs, orange) and gp41 MPER (cyan), respectively, that are preferentially expressed on functional Env trimers, whereas non-neutralizing Abs A32 and 7B2 target epitopes located in CD4-induced sites (CD4i conformation epitopes are not visible in the depicted pre-CD4 binding Env structure) and in the gp41 stalk (cyan), respectively, that are preferentially expressed on nonfunctional forms of Env. The depicted structure of Env trimer is derived from pdb 4NCO.
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ppat.1005233.g001: HIVxCD3 DARTs retarget cytolytic CD3+ T-cells to Env-expressing HIV-infected CD4+ T-cells.(A) Mechanism of cytolysis. The CD3 arm (orange) of the bi-specific DART binds to CD3 at the surface of CD3+ T-cells and the HIV arm (blue) binds to HIV Env at the surface of HIV-infected CD4+ T-cells. Cell surface Env may be in the form of functional mature trimers or nonfunctional variant forms such as cleaved or uncleaved gp160 monomers or gp41 stumps [43]. DART-mediated engagement of target and effector cells results in activation of effector cell cytolytic responses and target cell killing. (B) Variety of Env epitopes targeted by HIVxCD3 DARTs. Locations on the mature HIV-1 Env trimer surface of epitopes recognized by the anti-Env Abs used as sources of the HIV binding arms of DARTs are shown. Broadly neutralizing Abs PGT121, PGT145, VRC01 and 10E8 target epitopes located in the V3 glycan (N332; green), V2 glycan (N160K, blue), CD4 binding site (CD4bs, orange) and gp41 MPER (cyan), respectively, that are preferentially expressed on functional Env trimers, whereas non-neutralizing Abs A32 and 7B2 target epitopes located in CD4-induced sites (CD4i conformation epitopes are not visible in the depicted pre-CD4 binding Env structure) and in the gp41 stalk (cyan), respectively, that are preferentially expressed on nonfunctional forms of Env. The depicted structure of Env trimer is derived from pdb 4NCO.
Mentions: A series of bi-specific antibody constructs that bind simultaneously to HIV Env and human CD3 receptor were generated using the basic DART platform (Fig 1A) [29,30]. To maximize the breadth of Env recognition across multiple HIV isolates, complementarity determining regions (CDRs) from four bNAbs (PGT121, PGT145, 10E8, VRC01) [31–33] were incorporated into the Env-recognizing arm of HIVxCD3 DARTs. These bNAb-derived DARTs were compared with ones with HIV Env arms derived from two non-neutralizing antibodies (A32, 7B2) that bind broadly conserved residues in Env and efficiently induce antibody dependent cell-mediated cytotoxicity (ADCC) [34–41]. Each of the six designed bi-specific Abs recognizes a distinct epitope on the surface of HIV Env protein (Fig 1B). CDRs derived from palivizumab [42], an antibody recognizing the fusion protein of respiratory syncytial virus (RSV), were used to construct a negative control DART (RSVxCD3) that does not bind to HIV Env. The CD3-recognizing arm, which was identical in all DARTs, was derived from hXR32 [28], a humanized mouse anti-human CD3ε antibody, which cross-reacts with nonhuman primate CD3ε. The HIVxCD3 DARTs in basic format were produced by expression in stably transfected CHO cells and purified. The formation of properly assembled molecules was confirmed by reducing and non-reducing SDS-PAGE and analytical SEC; the average purity of the assembled HIVxCD3 DART molecules was 95%.

Bottom Line: In an ex vivo model using cells isolated from HIV-infected participants on cART, combinations of the most potent HIVxCD3 DARTs reduced HIV expression both in quiescent and activated peripheral blood mononuclear cell cultures isolated from HIV-infected participants on suppressive cART.Importantly, HIVxCD3 DARTs did not induce cell-to-cell virus spread in resting or activated CD4 T cell cultures.Collectively, these results provide support for further development of HIVxCD3 DARTs as a promising therapeutic strategy for targeting HIV reservoirs.

View Article: PubMed Central - PubMed

Affiliation: Gilead Sciences, Foster City, California, United States of America.

ABSTRACT
HIV reservoirs and production of viral antigens are not eliminated in chronically infected participants treated with combination antiretroviral therapy (cART). Novel therapeutic strategies aiming at viral reservoir elimination are needed to address chronic immune dysfunction and non-AIDS morbidities that exist despite effective cART. The HIV envelope protein (Env) is emerging as a highly specific viral target for therapeutic elimination of the persistent HIV-infected reservoirs via antibody-mediated cell killing. Dual-Affinity Re-Targeting (DART) molecules exhibit a distinct mechanism of action via binding the cell surface target antigen and simultaneously engaging CD3 on cytotoxic T lymphocytes (CTLs). We designed and evaluated Env-specific DARTs (HIVxCD3 DARTs) derived from known antibodies recognizing diverse Env epitopes with or without broadly neutralizing activity. HIVxCD3 DARTs derived from PGT121, PGT145, A32, and 7B2, but not VRC01 or 10E8 antibodies, mediated potent CTL-dependent killing of quiescent primary CD4 T cells infected with diverse HIV isolates. Similar killing activity was also observed with DARTs structurally modified for in vivo half-life extension. In an ex vivo model using cells isolated from HIV-infected participants on cART, combinations of the most potent HIVxCD3 DARTs reduced HIV expression both in quiescent and activated peripheral blood mononuclear cell cultures isolated from HIV-infected participants on suppressive cART. Importantly, HIVxCD3 DARTs did not induce cell-to-cell virus spread in resting or activated CD4 T cell cultures. Collectively, these results provide support for further development of HIVxCD3 DARTs as a promising therapeutic strategy for targeting HIV reservoirs.

No MeSH data available.


Related in: MedlinePlus