Development of tetravalent, bispecific CCR5 antibodies with antiviral activity against CCR5 monoclonal antibody-resistant HIV-1 strains.

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 (Gly4Ser)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 antibody-resistant HIV-1 strains.

Synthesis, SAR and evaluation of [1,4']-bipiperidinyl-4-yl-imidazolidin-2-one derivatives as novel CCR5 antagonists.

Elaboration of our previously disclosed spiropiperidine template led to the development of a series of novel CCR5 antagonists. Results of SAR exploration and preliminary lead characterization are described.

Novel hexahydropyrrolo[3,4-c]pyrrole CCR5 antagonists.

Starting with a high-throughput screening lead, a novel series of CCR5 antagonists was developed utilizing an information-based approach. Improvement of pharmacokinetic properties for the series was pursued by SAR exploration of the lead template. The synthesis, SAR and biological profiles of the series are described.

Spiropiperidine CCR5 antagonists.

A novel series of CCR5 antagonists has been identified, utilizing leads from high-throughput screening which were further modified based on insights from competitor molecules. Lead optimization was pursued by balancing opposing trends of metabolic stability and potency. Selective and potent analogs with good pharmacokinetic properties were successfully developed.

Molecular interactions of CCR5 with major classes of small-molecule anti-HIV CCR5 antagonists.

In addition to being an important receptor in leukocyte activation and mobilization, CCR5 is the essential coreceptor for human immunodeficiency virus (HIV). A large number of small-molecule CCR5 antagonists have been reported that show potent activities in blocking chemokine function and HIV entry. To facilitate the design and development of next generation CCR5 antagonists, docking models for major classes of CCR5 antagonists were created by using site-directed mutagenesis and CCR5 homology modeling. Five clinical candidates: maraviroc, vicriviroc, aplaviroc, TAK-779, and TAK-220 were used to establish the nature of the binding pocket in CCR5. Although the five antagonists are very different in structure, shape, and electrostatic potential, they were able to fit in the same binding pocket formed by the transmembrane (TM) domains of CCR5. It is noteworthy that each antagonist displayed a unique interaction profile with amino acids lining the pocket. Except for TAK-779, all antagonists showed strong interaction with Glu283 in TM 7 via their central basic nitrogen. The fully mapped binding pocket of CCR5 is being used for structure-based design and lead optimization of novel anti-HIV CCR5 inhibitors with improved potency and better resistance profile.

Novel CCR5 monoclonal antibodies with potent and broad-spectrum anti-HIV activities.

To identify monoclonal antibodies (mAbs) with high potency and novel recognition sites, more than 25,000 of mouse hybridomas were screened and 4 novel anti-human CCR5 mAbs ROAb12, ROAb13, ROAb14, and ROAb18 showing potent activity in cell-cell fusion (CCF) assay were identified. These mAbs demonstrated potent antiviral activities in both single-cycle HIV infection (IC(50) range: 0.16-4.3 microg/ml) and PBMC viral replication (IC(50) range: 0.02-0.04 microg/ml) assays. These potent antiviral effects were donor-independent. All 4 mAbs were also highly potent in the PhenoSense assay against 29 HIV isolates covering clade A through G. In all antiviral assays, these mAbs showed potency superior to the previously reported mAb 2D7 in side-by-side comparison studies. All 4 mAbs were also fully active against viruses resistant to HIV fusion inhibitor enfuvirtide and CCR5 antagonist maraviroc. Although ROAb12, ROAb14, and ROAb18 inhibited RANTES, MIP1alpha and MIP1beta binding and cell activation, the other novel mAb ROAb13 was inactive in inhibiting cell activation by these three ligands. Furthermore, highly synergistic antiviral effects were found between mAb ROAb13 and 2D7 or ROAb12. In addition, none of these mAbs showed agonist activity or caused internalization of the CCR5 receptor.

A study of the molecular mechanism of binding kinetics and long residence times of human CCR5 receptor small molecule allosteric ligands.

BACKGROUND AND PURPOSE: The human CCR5 receptor is a co-receptor for HIV-1 infection and a target for anti-viral therapy. A greater understanding of the binding kinetics of small molecule allosteric ligand interactions with CCR5 will lead to a better understanding of the binding process and may help discover new molecules that avoid resistance. EXPERIMENTAL APPROACH: Using [(3) H] maraviroc as a radioligand, a number of different binding protocols were employed in conjunction with simulations to determine rate constants, kinetic mechanism and mutant kinetic fingerprints for wild-type and mutant human CCR5 with maraviroc, aplaviroc and vicriviroc. KEY RESULTS: Kinetic characterization of maraviroc binding to the wild-type CCR5 was consistent with a two-step kinetic mechanism that involved an initial receptor-ligand complex (RA), which transitioned to a more stable complex, R'A, with at least a 13-fold increase in affinity. The dissociation rate from R'A, k-2 , was 1.2 × 10(-3) min(-1) . The maraviroc time-dependent transition was influenced by F85L, W86A, Y108A, I198A and Y251A mutations of CCR5. CONCLUSIONS AND IMPLICATIONS: The interaction between maraviroc and CCR5 proceeded according to a multi-step kinetic mechanism, whereby initial mass action binding and later reorganizations of the initial maraviroc-receptor complex lead to a complex with longer residence time. Site-directed mutagenesis identified a kinetic fingerprint of residues that affected the binding kinetics, leading to the conclusion that allosteric ligand binding to CCR5 involved the rearrangement of the binding site in a manner specific to each allosteric ligand.

The second extracellular loop of CCR5 contains the dominant epitopes for highly potent anti-human immunodeficiency virus monoclonal antibodies.

Six mouse anti-human CCR5 monoclonal antibodies (mAbs) that showed potent antiviral activities were identified from over 26,000 mouse hybridomas. The epitopes for these mAbs were determined by using various CCR5 mutants, including CCR5/CCR2B chimeras. One mAb, ROAb13, was found to bind to a linear epitope in the N terminus of CCR5. Strikingly, the other five mAbs bind to epitopes derived from extracellular loop 2 (ECL2). The three most potent mAbs, ROAb12, ROAb14, and ROAb18, require residues from both the N-terminal (Lys171 and Glu172) and C-terminal (Trp190) halves of ECL2 for binding; two other mAbs, ROAb10 and ROAb51, which also showed potent antiviral activities, require Lys171 and Glu172 but not Trp190 for binding. Binding of the control mAb 2D7 completely relies on Lys171 and Glu172. Unlike 2D7, the novel mAbs ROAb12, ROAb14, and ROAb18 do not bind to the linear peptide 2D7-2SK. In addition, all three mAbs bind to monkey CCR5 (with Arg at position 171 instead of Lys); however, 2D7 does not. Since five of the six most potent CCR5 mAbs derived from the same pool of immunized mice require ECL2 as epitopes, we hypothesize that CCR5 ECL2 contains the dominant epitopes for mAbs with potent antiviral activities. These dominant epitopes were found in CCR5 from multiple species and were detected in large proportions of the total cell surface CCR5. mAbs recognizing these epitopes also showed high binding affinity. A homology model of CCR5 was generated to aid in the interpretation of these dominant epitopes in ECL2.

CCR5 small-molecule antagonists and monoclonal antibodies exert potent synergistic antiviral effects by cobinding to the receptor.

A panel of four CCR5 monoclonal antibodies (mAbs) recognizing different epitopes on CCR5 was examined in CCR5-mediated cell-cell fusion assay, alone or in combination with a variety of small molecule CCR5 antagonists. Although no antagonism was observed between any of the CCR5 inhibitors, surprisingly potent synergy was observed between CCR5 mAbs and antagonists, and the synergistic activity was confirmed in other antiviral assays. Strong synergy was also observed between CCR5 inhibitors and the human immunodeficiency virus (HIV) fusion inhibitor enfuvirtide. There was no synergy observed between small molecule CCR5 inhibitors; however, potent synergy was observed between mAbs recognizing different parts of CCR5. In all synergistic combinations, greater synergy was achieved at higher percent inhibition levels. A negative correlation was found between the degree of synergy between the two classes of CCR5 inhibitors and the ability to compete each other for binding to the receptor. For example, the greatest synergy, observed between the mAb ROAb13 and the small molecule inhibitor maraviroc, did not interfere with binding to CCR5 for either inhibitor, whereas no synergy was found between mAb 45523 and maraviroc, which do compete for binding to CCR5. In addition, in contrast to a recent report, the CCR5 inhibitors tested here were found to inhibit the same stage of HIV entry. Based on the data presented here, we hypothesize that CCR5 inhibitors exert synergistic antiviral actions through a cobinding mechanism.

Analyzing ligand and small molecule binding activity of solubilized GPCRs using biosensor technology.

We used Biacore technology to measure directly the binding of natural ligands and small molecules to the chemokine receptors CXCR4 and CCR5. Both G protein-coupled receptors were solubilized from whole cell pellets and captured on antibody surfaces for analysis. Our solubilization conditions maintained high-affinity binding of chemokines SDF-1alpha and RANTES to CXCR4 and CCR5, respectively. Surface density- and buffer-dependent binding responses, along with binding data for a selective ligand (RCP-168), further validated the biosensor assay. In addition, we showed that it is possible to collect high-quality binding responses for the archetypal small molecule inhibitors JM-2987 and TAK-779. Finally, using our biosensor-based method, we characterized the kinetics of 19 novel small molecule inhibitors of CCR5 and showed that their affinities correlated with values determined for the membrane-associated receptor. Together, the chemokine and small molecule binding data provide evidence that the solubilized receptors maintain native binding properties. These solubilized receptor preparations could be useful reagents for biophysical studies as well as for structural analysis.