Software Technology for Erythrocyte-binding therapeutics

Invented by Jeffrey A. Hubbell, Stephan Kontos, Karen Y. Dane, Ecole Polytechnique Federale de Lausanne EPFL

The Ecole Polytechnique Federale de Lausanne EPFL invention works as follows

Background for Erythrocyte-binding therapeutics

Rejection of transplanted tissues and autoimmune diseases are two types of pathological conditions that result in immunorejection due to an antigenic biomolecule. Immunorejection can be suppressed or treated with many drugs and clinical procedures. Vaccines capitalize on this by stimulating the immune response to antigens from pathogenic biomolecules in order to increase the immune system’s response against the biomolecules or proteins that contain the antigen.

Tolerogenesis refers to the process of creating an immune tolerance to a substance. Patients, whether human or non-humans, who are treated to make tolerance to a substance will have a decreased adapative immunity response to that substance. Analyzing the amount of antibodies that are circulating to the substance or T-cell reactions to it can determine if there has been a reduction in adaptive immune responses. These compositions and methods of tolerization can be found here. Many embodiments require administration of a fusion moiety that contains a tolerizing and erythrocyte binding moiety. The fusion molecule binds with erythrocytes, initiating a process to present the tolerizing antibody to the immune system in a way that creates tolerance.

Peptides that specifically bind only to erythrocytes (also called red blood cells) were discovered. These peptide-binding moieties bind to erythrocytes regardless of the presence of blood factors. These ligands can be used in many ways.

An embodiment is a composition that is pharmaceutically acceptable and contains a tolerogenic agent and an erythrocyte binding moiety. The erythrocyte binding moiety binds specifically to the biomolecule selected from the following group: Band 3 (CD233), Aquaporin-1 (CD240CE), Rh30CE(CD240CE), Rh30CE) and Rh30CE (CD240CE), Kidd antigen, RhAg/Rh50, RhAg/Rh50, CD235CE), glycophorin (CD235CE), glycophorin (CD235CE), glycophorin (CD235CE), CD235CE), glycophorin (CD235CE), CD235CE), glycophorin-binding (CD235C4A/B-CAM-4 (CD235CE), erythrocyte-binding, stomatin), stomatin (CD123), erythronic antigen), CD240CE), CD240CE (CD240CE), erythrocytestom), erythrocytes), erythrocyte-CD240CE), erythrocyte-binding to erythrocytes), erythronic antigen (CD235CE), ), stom), erythrocyte-stom), stom), erythrocyte-binding to the erythrocytes), erythrocytes), erythrocytes (CD28), erythrocytes), erythrocytes (CD123), erythrocytes), erythrocyte (CD210CE), erythrocyte (CD240CE), and erythrocyte (CD235CE), and erythrocyte (CD240CE), and erythrocyte (CD235CE), which erythrcell-binding to the erythrocyte (CD123), to the patient’sto (CD139), and erythrocyte-binding (CD139), and erythrocyte (CD139), and erythrocyte (CD235CE), and erythrocyte (CD231)), and erythrocyte (CD235CE), and erythrocyte (CD123), and erythr

An embodiment is a composition that is pharmaceutically acceptable and comprises: an erythrocyte binding moiety linked to a domain that binds to a target, e.g. a protein that contains a tolerogenic antibody. These domains can be either a peptidic or an antibody ligand, or a fragment of an antibody.

Another embodiment” is a composition that can be used to remove antibodies from the bloodstream. The composition contains an erythrocyte binding moiety that is joined to an antigen. This could be a native antigen or an antigen for therapeutic proteins.

Tolerogenesis is possible through the use of molecular designs. A conjugate is a protein or molecular antigen that is to be tolerated. It is formed by an erythrocyte binding component. A peptide ligand or antibody, an aptamer, a fragment, or a moiety can make up the moiety. A molecular fusion is also known as a conjugate. It may contain a fusion protein, an antibody, or an aptamer.

Here are described peptides that bind specifically to erythrocytes. These peptides can be provided as peptidic-ligands with specific sequences or antibodies or fragments thereof that bind to erythrocytes. These peptides can be prepared by molecular fusions of therapeutic agents or targeting peptides. When therapeutic agents are part of a fusion, they may have a longer half-life in vivo. The fusions may result in immunotolerance. In this context, the term “antigen” refers to the entire antigen or an antigenic fragment of it, as well as a mimetic. Fusions using targeting peptides direct fusions to the target. For example, a tumor. The erythrocyte binding ligands recruit erythrocytes towards the target to reduce or eliminate blood flow to that tumor.

Peptidic Sequences That Specifically Bind Erythrocytes.

Peptidic binding ligands were found to be able to bind to the erythrocyte cells surfaces, without altering their morphology or cytoplasmic translocation. The ligands were distributed evenly across the cell surface without clustering. Glycophorin A (GYPA), was identified as ERY-1’s target protein. ERY-1 was only reactive with rat and mouse species. It was found that peptididic ligands specifically bound humanerythrocytes. As shown in Tables 1 and 2, other experiments revealed binding ligands that could be used to bind human erythrocytes. Six sequences were specifically bound to human erythrocytes. A seventh sequence, named ERY50, bound human erythrocytes and also bound epithelial/endothelial cells.

TABLE?1\nPeptidic?ligands?that?bind?human?erythrocytes\nPeptide Human?Erythrocyte?Binding Sequence\nName Peptide?Sequence Identifier\nERY19 GQSGQ PNSRWIYMTPLSPGIYR GSSGGS SEQ?ID?NO:?4\nERY50 GQSGQ SWSRAILPLFKIQPV GSSGGS SEQ?ID?NO:?5\nERY59 GQSGQ YICTSAGFGEYCFID GSSGGS SEQ?ID?NO:?6\nERY64 GQSGQ TYFCTPTLLGQYCSV GSSGGS SEQ?ID?NO:?7\nERY123 GQSG HWHCQGPFANWV GSSGGS SEQ?ID?NO:?8\nERY141 GQSGQ FCTVIYNTYTCVPSS GSSGGS SEQ?ID?NO:?9\nERY162 GQSGQ SVWYSSRGNPLRCTG GSSGGS SEQ?ID?NO:?10\nUnderlined sequence portions indicate linker sequences

TABLE?2\nPeptidic?ligands?that?bind?mouse?or?human\nerythrocytes\nPeptide Sequence?Identifier\nERY19? PNSRWIYMTPLSPGIYR SEQ?ID?NO:?11\nERY50? * SWSRAILPLFKIQPV SEQ?ID?NO:?12\nERY59? YICTSAGFGEYCFID SEQ?ID?NO:?13\nERY64? TYFCTPTLLGQYCSV SEQ?ID?NO:?14\nERY123? HWHCQGPFANWV SEQ?ID?NO:?15\nERY141? FCTVIYNTYTCVPSS SEQ?ID?NO:?16\nERY162? SVWYSSRGNPLRCTG SEQ?ID?NO:?17\nERY1** WMVLPWLPGTLD SEQ?ID?NO:?1\n*not specific for erythrocytes\n**for mouse

The invention includes peptides that specifically bind to the surface of erythrocytes. Sequences were not optimized to be as short as possible. This optimization is possible with the techniques described in this article. For example, Kenrick et al. (Protein Eng. Des. Sel. (2010) 23(1):9-17). Screened from a library of 15 residues, we then identified 7 minimal binding sequences. Getz (ACS Chem. Biol., May 26, 2011, identified minimal binding domains of as little as 5 residues length. There may be multiple erythrocyte binding proteins in the same sequences. For example, there could be between 2 and 20 repeats. Craftsmen will quickly appreciate that all values and ranges within the stated ranges are possible. The peptides can also be combined, meaning that two or more sequences could be in the same peptide, or part of a single molecular combination.

The number of residues providing specific binding in a sequence is between 4 and 12. All peptides with four consecutive residues are listed in Table 2. This number is calculated based on the number residues for other peptidic-protein-binding ligands. The invention includes minimum length sequences that correspond to one of the erythrocyte binding SEQ IDs, as shown in Table 1. Certain embodiments may also include a composition that contains a peptide or an isolated (or purified peptide) of an amino acid sequence. These sequences can be chosen from the following groups: SEQID NO:11; SEQID NO:13; SEQID NO:14; SEQID NO:15. SEQID NO:16. SEQID NO:17. SEQID NO:1 and conservative substitutions thereof. The sequence binds to an erythrocyte. Alternately, the number of consecutive residues can be set to between 5 and 18. Craftsmen will quickly appreciate that all values and ranges within the stated ranges are possible, e.g. 7, 8, 9, 10, 8 or 18. You may find the erythrocyte binding sequence to have, for example, a conservative substitution of at most one but not more than two of the sequences or 1, 2, 3 or 3 substitutions or 1 to 5 substitutions. It is possible to substitute L-amino acid in the sequence discovered with D-amino acid, as shown in Giordano. In some embodiments, the peptide/composition may consist of a sequence selected from the following groups: SEQID NO:11, 13, 14, 15 and 15, SEQID NO:15 and 16, SEQID NO:16, 17 and 18. Although the peptide might be short in length (e.g. having between 10 and 100 residues), artisans will quickly realize that all values and ranges within the group consisting of SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, and SEQID No.15 are possible. An erythrocyte binding moiety of peptides may be included that contains a peptide-ligand with a dissociation constant between about 10 mM and 0.01 nM. This is determined from equilibrium binding measurements between the protein and the erythrocytes. Artists will quickly appreciate that all values and ranges within the stated ranges are possible, e.g. 1?M to 1 nM. A therapeutic agent may also be contained in the peptide. A therapeutic agent could be a protein, biologic, an antibody fragment or a peptide. A tolerogenic antigen may also be included in the peptide, such as a human protein that is used in treating a person who is deficient in it (e.g. blood factors like factor VIII or factor IX), synthetic proteins not found in humans, human food allergens or human autoimmune antibodies).

Polypeptides can be of different lengths depending on the application. Polypeptides that have the polypeptide-ligand sequences will show specific binding if they are available for interaction in vivo with erythrocytes. These methods can be used to test peptides that are likely to fold. Certain embodiments address polypeptides with a polypeptide-ligand, but not in nature. Other embodiments target polypeptides of particular lengths. For example, 6 to 3000 residues or 12 1000 or 12-100 or 10-50. Craftsmen will quickly appreciate that all values and ranges within these limits are possible.

Certain embodiments offer different polypeptide sequences, purified or isolated polypeptides, and/or both. A polypeptide refers to an amino acid chain that is free from post-translational modifications (e.g. phosphorylation, glycosylation, and/or complexation, synthesis into multisubunit compounds, with nucleic acid and/or carbohydrate, or any other molecules. Proteoglycans are also referred to as polypeptides. A?functional polypeptide’ is as it is used herein. A polypeptide capable of performing the specified function. There are many ways to make polypeptides, some of which are well-known in the art. Polypeptides can be made by a variety of methods, including extraction from isolated cells, expression of a recombinant DNA encoding the polypeptide or chemical synthesis. For example, polypeptides can also be made using recombinant technology and expression vectors that encode the polypeptide are introduced into host cells (e.g. by transformation or transfection) to allow for the expression of the encoded protein.

There are many conservative modifications that can be made to an amino-acid sequence without affecting its activity. These modifications are called conservative substitutions or mutants. This means that an amino acid from a group of amino acids with a certain size or characteristic can be replaced by another amino acid. You can choose from other members in the same class as the amino acid to substitute for an amino acids sequence. The nonpolar (hydrophobic), amino acids are alanine and leucine. Glycine, serine and threonine are the polar neutral amino acid. Histidine, lysine, and arginine are the positively charged (basic), amino acids. Aspartic acid, glutamic acid, and other negatively charged amino acids are the acidic. These alterations will not have a significant effect on the apparent molecular weight, as determined by polyacrylamide Gel Electrophoresis and isoelectric point. You can also substitute optical isomers for sequences, such as D amino acids replacing L amino acids. This is a conservative substitution. A D to L substitution may be performed on all amino acids within a sequence. Examples of conservative substitutions are Lys for Asp and vice-versa to keep a positive charge, Glu for Asp to keep a negative charge, Ser for Thr to preserve a free?OH, and Gln For Asn to preserve a free N2. In some cases, substitutions can be made with no loss of function, such as point mutations, deletions or insertions of nucleic acids sequences or polypeptide sequences. Substitutions can include, for example, 1, 2, 3 or more residues. These amino acid residues can be described using either the single-letter amino acid designationator or the three letter abbreviation. These abbreviations are consistent with J. Biol.’s standard polypeptide nomenclature. Chem., (1969), 243, 3552-3559. All sequences of amino acids are represented in formulae with left or right orientations in the traditional direction from amino-terminus through carboxy-terminus.

In certain cases, it may be necessary to determine the percent identity of a protein to a sequence described herein. The percent identity of a peptide to a sequence is sometimes required. In these cases, it is determined by the number of residues or a portion. A polypeptide with a 90% identity may also be a part of a larger protein.

Polypeptides can include a chemical modification. This term, in this context refers to a change or addition in the chemical structure of naturally occurring amino acids. These modifications can be made to a sidechain or a terminus. For example, changing the amino terminus or carboxyl terminus. The modifications can be useful in creating chemical groups that can be used to link polypeptides with other materials or attach a therapeutic agent.