CCR5-specific antibodies with HIV neutralizing activity that recognize a linear dodecapeptide that mimics a conformational epitope in the second extracellular loop of CCR5

#This invention relates, e.g., to peptides which closely mimic conformational epitopes recognized by monoclonal antibodies of interest, to nucleic acids encoding the peptides, and to monoclonal and polyclonal antibodies specific for the peptides.

#Infectivity of certain viruses, particularly HIV and related viruses, such as SIV, SHIV and other immune deficiency viruses, is mediated by the co-receptor, CCR5. This co-receptor has been identified as a major target for HIV-1 entry inhibitors, since most of the viruses responsible for person to person transmission have been typed as CCR5-using (R5) strains. The naturally occurring Δ32 ccr5 allele, when homozygous, is associated with resistance to in vitro infection of CD4+ cells with R5 viruses. Moreover, Δ32 ccr5 homozygosity confers considerable protection against HIV infection in vivo. Yet this genotype is not associated with abnormal immune function, and may be dispensable due to redundancy in chemokine receptor usage.

#Three main classes of CCR5-targeting inhibitors have been reported: CC-chemokine analogues, small molecules, and monoclonal antibodies. One of the most active monoclonal antibodies targeting CCR5 is mAb 2D7, which was generated from the spleen of C57BL/6 mice immunized with the murine pre-B cell lymphoma line L1.2, which expresses high levels of transfected CCR5. This murine antibody was shown to inhibit in vitro infections of CD4+CCR5+ human cells by most R5-tropic viruses at an ID50 of 2-10 μg/ml, making it a good candidate for generating humanized antibodies. However, no success in humanizing this mAb has been reported. The epitope recognized by mAb 2D7 on CCR5 has been partially mapped to the first half of the second extracellular loop (ECL-2) by mutagenesis studies. Amino acids 171-KE-172 were found to be critical for mAb 2D7 binding. But the epitope was determined to be conformation-dependent, and the binding is lost in CCR5 mutants lacking the disulfide bridge between ECL-1 and ECL-2, as well as in reduced forms of CCR5 extracted from cells with various detergents.

#FIG. 1 shows that selected phage clones react specifically with 2D7 in phage ELISA. Selection of phages expressing random 12-mer peptides was performed on mAb 2D7-coated plates. Clones p2D7-1SK through p2D7-5SK were eluted from panning round IV and were analyzed for their binding to mAb 2D7 by phage ELISA as described in Example I.

#FIG. 2 shows schematically the conformational structure of the 2D7 epitope on CCR5. The peptide sequences of four specific 2D7-binding phage clones (FIG. 1) from panning round IV were identical. Alignment of the phage-displayed peptide motif with CCR5 ECL-2 identified two regions of homology. These regions of homology are the underlined portions of the 2D7-2SK peptide sequence, E-W-Q-K-E-G-L-V-T-L-W-L (SEQ ID NO:1); the underlined Q-K-E-G-L segment is SEQ ID NO:2. The thread model of human CCR5 is adapted from Siciliano et al (1999) J Biol Chem 274, 1905-13.

#FIG. 3 shows the characterization of mAb 2D7 binding to synthetic peptide 2D7-2SK peptide by ELISA. FIG. 3A shows direct binding of mAb 2D7 to different concentrations of biotinylated-2D7-2SK or to biotinylated-scrambled 2D7-2SK peptide (2D7-2SK-SCR) captured by streptavidin-coated microtiter plates. The bound antibodies were quantified by HRP-conjugated goat anti-mouse IgG antibody. FIG. 3B shows competition of binding of phages displaying peptide 2D7-2SK sequence (p2D7-2SK) to mAb 2D7 by soluble 2D7-2SK synthetic peptide. Microtiter wells, coated with mAb 2D7 at 200 ng/well, were incubated with serially diluted synthetic 2D7-2SK peptide or with a control peptide (CGRAARIGFPGAYTTKNG) (SEQ ID NO:3) for 30 min at room temperature, p2D7-2SK phages were then added to all wells (109 phages/well), followed by addition of HRP-conjugated anti-phage antibodies. FIG. 3C shows the identification of critical residues in the 2D7-2SK peptide sequence required for mAb 2D7 binding. MAb 2D7 (50 ng/100 μl/well) was added to plates coated with either unmodified biotin-2D7-2SK peptide or mutated biotin-2D7-2SK peptide derivatives (Table 1), all captured on streptavidin coated wells. The bound 2D7 antibodies were quantified by HRP-conjugated goat anti-mouse IgG antibody. The absorbance value (0.49) of mAb 2D7 binding to 2D7-2SK peptide (unmodified) is represented as 100% control binding.

#The inventors describe herein methods for identifying peptides which closely mimic conformational epitopes recognized by neutralizing monoclonal antibodies that bind to a pathogen or to a receptor or co-receptor on a cell targeted by the pathogen. In particular, methods are described for identifying peptides which closely mimic conformational epitopes recognized by a neutralizing monoclonal antibody (mAb) that binds to the HIV co-receptor CCR5 (e.g., the murine mAb 2D7). Briefly, the method comprises screening (panning) a random peptide library (such as a peptide phage display library) for peptides that bind preferentially and with high affinity (avidity) to a mAb of interest (e.g., 2D7). In one embodiment, the panning is performed in culture medium, such as EMEM (Eagle's Modified Eagles Medium) supplemented with serum (e.g., FBS). This differs from more conventional panning procedures, in which the panning is performed in a buffer, such as PBS. In another embodiment, the panning is performed in a buffer or in culture medium; then candidate peptides are further screened with one or more relevant biological assays, such as the ability of the peptide to inhibit antibody blocking of viral fusion (syncytia formation) or entry of the virus into a target cell. Following the panning and optional further screening, a candidate peptide is tested for its ability to produce a neutralizing antibody (such as a human antibody) against HIV (or SIV, SHIV, etc.), or against another pathogen of interest. One aspect of the invention is a neutralizing antibody produced by a method as above.

#Example II shows that a peptide (represented by SEQ ID NO:1) identified by a method of the invention, which closely mimics a conformational epitope of CCR5, binds to mAb 2D7 with high affinity and can significantly reduce 2D7's ability to bind to CCR5 and block HIV-1 fusion. This peptide was conjugated to the adjuvant KLH and used to immunize rabbits, thereby generating polyclonal antibodies that bound to the HIV co-receptor and blocked HIV fusion and infection of target cells, including peripheral blood lymphocytes, from human and monkeys.

#Another aspect of the invention is an isolated peptide comprising a sequence that is at least about 60% identical (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100% identical) to the sequence E-W-Q-K-E-G-L-V-T-L-W-L (Glu-Trp-Gln-Lys-Glu-Gly-Leu-Val-Thr-Leu-Trp-Leu) (SEQ ID NO:1) over its entire length. That is, the contiguous amino acid residues of that sequence of the peptide are at least about 60% identical to the contiguous amino acids of SEQ ID NO:1. For example, the peptide may have about 1-3 internally located amino acid insertions, deletions or substitutions compared to SEQ ID NO:1 and/or the peptide may lack about 1-3 amino acids from one of both termini of SEQ ID NO:1. Another aspect of the invention is an isolated peptide which is an active variant of a peptide comprising SEQ ID NO:1. For example, the peptide may have about 1-3 internally located amino acid insertions, deletions or substitutions compared to SEQ ID NO:1 and/or the peptide may lack about 1-3 amino acids from one of both termini of SEQ ID NO:1. In one embodiment, a peptide comprising a sequence that is about 60% identical to SEQ ID NO:1 is limited in size, e.g. it is no more than about 100, 80, 60, 50, 40, 30 or 20 amino acids in length. In another embodiment, the isolated peptide consists of SEQ ID NO:1, or consists of a sequence that is an active variant of SEQ ID NO:1 and/or is at least about 60% identical (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100% identical) to the contiguous amino acid sequence of SEQ ID NO:1. Embodiments of the invention include peptides that differ by 1 amino acid from SEQ ID NO:1 (are about 92% identical to it) or that differ by 2 amino acids (are about 83% identical); by 3 amino acids (about 75% identical); by 4 amino acids (about 67% identical) or by 5 amino acids (about 60% identical). An active variant peptide of the invention (e.g. a peptide that exhibits the % identity to SEQ ID NO:1 discussed above) will maintain a biological function exhibited by a peptide comprising SEQ ID NO:1. For example, the variant peptide may bind preferentially and with high affinity to a mAb that binds to a conformational epitope of CCR5 that is involved in HIV, SHIV or SIV infection; and/or it may bind preferentially and with high affinity to mAb 2D7.