Peptides and Mimotopes

Links of interest: Mimo DB

Definitions

Anti-Idiotype antibody: binds to the idiotype of another antibody, usually an antibody drug.

Idiotype: is the sepcific combination of idiotopes present within an an antibodies CDRs. A single idiotope, is a specific region within an antibodies Fv region which binds to the paratope (antigenic epitope binding site) of a different antibody. Thus, an idiotope can be considered almost synonymous with an antigenic determinant of an antibody.

Mimotopes: are peptides with affinities to given targets. they are reaily obtained through biopanning against combinatorial peptide libraries constructed by phage display and other display technologies such as mRNA display, ribosome display, bacterial display and yeast display. Mimotopes have been used to infer the protein interation sites and for developing new diagnostics, therapeutics and vaccines. (Humang “MimoDB 2.0: a mimotope database and beyond” Nucleic Acids Research, 2012, 40, D271-D277 (November 2011).

A mimetope is a determinant which is recognized by the same binding molecule, such as an antibody, as a particular “eptiope” but which has a different composition form the “epitope”. For example, a binding molecule can be an antibody which recognizes (i.e., bind to) and eptiope comprising a linear sequence of amino acids. A “mimetope” of this epitope comprises a different linear sequen of amino acids but which is still recognzied by the same antibody. ( Messmer, US Patent Application No: 15/944,099, published as US 2018/0291059)

Phage Display of Peptides

Phage display of foreign peptides is an established technique routinely used. Phage peptide libaries can be constructed by fusing DNA containing a degenerate region to a gene encoding a coat protein usually gene III or gene VIII. This allows the peptide to be expressed as an N or C terminal fusion on the surface of the M13 bacteriophage (page) coat protein. A large number of random peptide libraries displayed on bacteriophage are now available, some are disulfide constrained by inerting two cystein residues. A typical library size ranges from 6-45 amino acid residues. Selection of peptides of interest from the library that bind to a target molecule can then be performed by “panning”. (Casey, “Phage display of peptides in ligand selection for use in affinity chromatography”, Methods in Molecular Biology, 421, Affinity Chromatography, Methods and Protocls, Second Edition. )

Identification of peptide mimotope ligands for antibodies:

Messmer (“Identification of peptide mimotope ligands for natalizumab” Scientific Reprots, 8, 14473 (2018) discloses the use of phage display libaries to identify peptide ligands they call Veritopes™ that bind natalizumab, a therapeutic mAb indicated for use in multiple sclerosis. PKNPSKF was identified as a novel natalizumab-binding motif and peptides containing this motif demonstrated utility as capture reagents in ELISAS. A peptide containing the motif was also shown to be competitive with the natural ligand (alpha4-integrin) and a neutralizing anti-idiotype antibody for natalizumab binding.

Peptides and Mimotopes for Affinity chromatoraphy

Peptide ligand affinity chromatography:

The advantages of peptide ligands for use in affinity chromatography include the relative low cost of high quality stables peptides and instead of having to preapre an affinity column using the whole recombinant antien, peptides that represent, for example, the antibody-binding site can be used. (Casey, “Phage display of peptides in ligand selection for use in affinity chromatography”, Methods in Molecular Biology, 421, Affinity Chromatography, Methods and Protocls, Second Edition. )

Messmer (US Patent Application No: 15/944,099, published as US 2018/0291059) discloses slecting mimetope peptides from phage display libraries , some of which contain cysteines flanking the peptide mimetope sequence to increase stability of the peptide through disulfide bonding formation. After three rounds of election with multiple phage display libaries, indiviudal phage plaques are isolated and sequenced. All clones are individually amplified, purified and their ability to specifically bind natalizumab-coated wells were assesed. The phage clone demonstrating specific, but low affinity binding to natalizumab is chemically synthesized with an N-temrinal acetyl modificaiton and a disulfide bridge between cysteins 2 and 1-0 or cysteins 8 and 16. The mimeotpe can be immobilzied onto beads and used for purificaiton of natalizumab.

Yang (J. Chromatography A, 1216 (2009) 910-918) disclose the use of hexamer peptides (HWRGWV, HYFKFD and HFRRHL) to selectively absorbe and isoalte human IgG.

Peptide mimotopes:

Casey, (“Phage display of peptides in ligand selection for use in affinity chromatography”, Methods in Molecular Biology, 421, Affinity Chromatography, Methods and Protocls, Second Edition. ) discloses methods to isolate a peptide mimotope from a pahge displayed random peptide library by isolation of a peptide that can mimic the sahpe of the antigen epitope and can be used to select antibodies that bind to this particular egion of the antigen. Casey also describes a process of purifying antibodies from human serum that bind to this peptide mimic.

Jensen (J of Immunological Methods, 284 (2004) 45-54) describes efficient purificaiton of unique antibodies using peptide affinity-matrix columns. Phage display was used to identify peptide ligands with unique specificity for mAbs. One peptide was linked to beaded agarose and demonstrated excellent performance as a peptide affintiy chromatogrpahy amtrix.

Sahin (WO 2017/008844) discloses peptide mimotopes of the CD3 T cell co-receptor epsilon chain and uses of the mimotope for anti-CD3alpha antibody purificaiton.

Simith (J of Chromatography B, 766 (2001) 13-26) describe a polyvalent, lytic pahge display system displaying a random peptide library to discover novel mimotopes reactive with a therapeutic mAb. The novel synethic peptide was linked to beaded agarose and the performance as a mimotope affinity chromatogrpahy matrix was evaluated.

Complications

Although pahge display libraries provide a huge resource for eptiope analysis and identificaiton of mimotopes, using the sequences derived directly as a useful affinity ligands for antibody purificaiton purposes is not trivial. This seems to be due, at least in part, to the fact that the phage derived sequences have relatively low affinity and the reaction kinetics are such that binding of the antibody to the ligand cannot occur during the short time that the antibody is in contact with the matrix. (Murray, Analytical Biochemistry 296, 9-17 (2001).

Coupling to the resin

Mimope peptide and carrier protein conjugates have been synthesized as coating antigens in immunoassay via the covalent coupling of amino or carboxyl groups of the peptide and the carrierty protein by using an active ester or by applying gltaraldehyde methods. However, these chemical coupling methods likely block the active side of mimotpe peptides, resulting in the loss or alteration of mimicking activities. (Zu, Talanta 146 (2016) 394-400).

Abtides

Abtides refers to peptides that mimic the binding specificity of a larger molecule such as an antibody or receptor.

Alvarex (US 5,885,577) discloses a process of identifying abtides by screening peptide libraries in a two-step prcoess. In the first screening step an antibody or receptor was used as the first target ligand. This step identifies peptide sequences termed “epitopes” or “mimetopes” which specifically bind the target ligand. An eptiope or mimetope is then used as a second target ligand in a second screening step to idenity a peptide sequence that are known as “abtides”.