CD4-anchoring HIV-1 Fusion Inhibitor with Enhanced Potency and in Vivo Stability

The development of an anti-HIV microbicide is critical in the fight against the spread of HIV. It is shown here that the covalent linking of compounds that bind gp120 with compounds that bind gp41 can inhibit HIV entry even more potently than individual inhibitors or noncovalent combinations. The most striking example involves griffithsin, a potent HIV inhibitor that binds to the surface of HIV gp120. While griffithsin inhibits HIV Env-mediated fusion in a CCR5-tropic cell-cell fusion assay with a 50% inhibitory concentration (IC 50 ) of 1.31 ؎ 0.87 nM and the gp41-binding peptide C37 shows an IC 50 of 18.2 ؎ 7.6 nM, the covalently linked combination of griffithsin with C37 (Griff37) has an IC 50 of 0.15 ؎ 0.05 nM, exhibiting a potency 8.7-fold greater than that of griffithsin alone. Similarly, in CXCR4-tropic cell-cell fusion assays, Griff37 is 5.2-fold more potent than griffithsin alone. In viral assays, both griffithsin and Griff37 inhibit HIV replication at midpicomolar levels, but the linked compound Griff37 is severalfold more potent than griffithsin alone against both CCR5-and CXCR4-tropic virus strains. Another example of this strategy is the covalently linked combination of peptide C37 with a variant of the gp120-binding peptide CD4M33 (L. Martin et al., Nat. Biotechnol. 21:71-76, 2003). Also, nuclear magnetic resonance (NMR) spectra for several of these compounds are shown, including, to our knowledge, the first published NMR spectrum for griffithsin.

Section: Discussionmentioning

confidence: 99%

Potent Strategy To Inhibit HIV-1 by Binding both gp120 and gp41

Kagiampakis

Gharibi

Mankowski

et al. 2011

“…Resistance via these routes could be prevented by the introduction of a second, independent pharmacophore. We recently described bifunctional HIV fusion inhibitor (BFFI) molecules as a new class of HIV entry inhibitors where a fusion inhibitor is physically linked to either an anti-CCR5 or anti-CD4 antibody (17,19,21). A similar linkage of a fusion inhibitor to one of the tetravalent CCR5 antibodies described here would combine three different pharmacophores in one molecule and should greatly increase the barrier to resistance development.…”

Section: Vol 55 2011mentioning

confidence: 99%

Development of Tetravalent, Bispecific CCR5 Antibodies with Antiviral Activity against CCR5 Monoclonal Antibody-Resistant HIV-1 Strains

Schanzer

Jekle

Nezu

et al. 2011

Self Cite

In this study, we describe novel tetravalent, bispecific antibody derivatives that bind two different epitopes on the HIV coreceptor CCR5. The basic protein formats that we applied were derived from Morrison-type bispecific antibodies: whole IgGs to which we connected single-chain antibodies (scFvs) via (Gly 4 Ser) n sequences at either the C or N terminus of the light chain or heavy chain. By design optimization, including disulfide stabilization of scFvs or introduction of 30-amino-acid linkers, stable molecules could be obtained in amounts that were within the same range as or no less than 4-fold lower than those observed with monoclonal antibodies in transient expression assays. In contrast to monospecific CCR5 antibodies, bispecific antibody derivatives block two alternative docking sites of CCR5-tropic HIV strains on the CCR5 coreceptor. Consequently, these molecules showed 18-to 57-fold increased antiviral activities compared to the parent antibodies. Most importantly, one prototypic tetravalent CCR5 antibody had antiviral activity against virus strains resistant to the single parental antibodies. In summary, physical linkage of two CCR5 antibodies targeting different epitopes on the HIV coreceptor CCR5 resulted in tetravalent, bispecific antibodies with enhanced antiviral potency against wild-type and CCR5 antibodyresistant HIV-1 strains.

“…Based on this concept, multiple domain binding CCR5 antibodies, such as PRO 140 (formerly known as PA14) (23) or 45549 (18) or bispecific antibodies, which have been proposed for cancer therapy (4), should be more resilient to this resistance mechanism. Alternatively, antibodies that are coupled to a second, independent pharmacophore, such as a fusion inhibitor (12,14,15), can also provide a higher barrier to resistance.…”

Section: Discussionmentioning

confidence: 99%

Epitope Switching as a Novel Escape Mechanism of HIV to CCR5 Monoclonal Antibodies

Jekle

Chhabra

Lochner

et al. 2010

Self Cite

In passaging experiments, we isolated HIV strains resistant to MAb3952, a chemokine (C-C motif) receptor 5 (CCR5) monoclonal antibody (MAb) that binds to the second extracellular domain (extracellular loop 2 [ECL-2]) of CCR5. MAb3952-resistant viruses remain CCR5-tropic and are cross-resistant to a second ECL-2-specific antibody. Surprisingly, MAb3952-resistant viruses were more susceptible to RoAb13, a CCR5 antibody binding to the N terminus of CCR5. Using CCR5 receptor mutants, we show that MAb3952-resistant virus strains preferentially use the N terminus of CCR5, while the wild-type viruses preferentially use ECL-2. We propose this switch in the CCR5 binding site as a novel mechanism of HIV resistance.HIV infects host cells by attaching to the CD4 receptor and subsequently binding to one of two major coreceptors, chemokine (C-C motif) receptor 5 (CCR5) or chemokine (C-X-C motif) receptor 4 (CXCR4). Both coreceptors are members of the superfamily of G-protein-coupled receptors, which are characterized by seven transmembrane helices, three extracellular loops, an amino-terminal domain (NTD), and an intracellular domain (2, 3). In CCR5, the two major binding regions for the viral gp120 envelope protein (Env) are the NTD and extracellular loop 2 (ECL-2) (8,9,26).Individuals who are homozygous for the CCR5 ⌬32 deletion have a natural resistance to HIV-1 infection, while heterozygous individuals have a reduced rate of infection; homozygous CCR5 ⌬32 individuals are healthy and immunocompetent, implying that normal CCR5 may be dispensable (7,19,27). This makes CCR5 an attractive target for antiretroviral drug development. The U.S. Food and Drug Administration approved maraviroc (Selzentry) in 2007 as the first small-molecule CCR5 inhibitor (20). Other CCR5 inhibitors, both small molecules (vicriviroc, aplaviroc, INC9B47, and others) and anti-CCR5 monoclonal antibodies (CCR5 MAb) (HGS004 and Pro-140) are or have been in clinical development (11, 16, 17, 22; E.