Methods for determining influence of carrier peptide on the binding of a therapeutic protein to a cell membrane

Abstract

The present application is concerns methods of measuring the influence of carrier peptides on cell binding of a therapeutic protein, particularly, botulinum toxin Type A, 150 kD. Carrier peptides and their uses are also described.

Description

METHODS FOR DETERMINING INFLUENCE OF CARRIER PEPTIDE ON THEBINDING OF A THERAPEUTIC PROTEIN TO A CELL MEMBRANEPRIORITY CLAIM

[0001] This International application is based upon and claims benefit of priority to US Provisional Application No. 63 / 584,901, which was filed on September 25, 2023 and is hereby incorporated by reference in its entirety.SEQUENCE LISTING

[0002] The Sequence Listing submitted herewith as a xml file named “13720.0038P1. xml” created on September 25, 2024, and having a size of 21 Kilo Bytes is hereby incorporated by reference pursuant to 37 C.F.R § 152€(5).FIELD OF THE INVENTION

[0004] This invention relates to methods of measuring the influence of carrier peptides on cell binding of a therapeutic protein, particularly, botulinum toxin Type A, 150 kD. Carrier peptides and their uses are also described.BACKGROUND OF THE INVENTION

[0005] Therapeutic proteins that are subcutaneously administered can still need to be taken into a cell to have the intended therapeutic effect. Peptide carriers are accessory excipients that can facilite the uptake of the therapeutic protein into a cell. Assays that mesure the influence of peptide carriers on the uptake of a therapeutic protein into a cell can be useful.SUMMARY OF THE INVENTION

[0006] The present disclosure is directed to methods for determining influence of carrier peptide on the binding of a therapeutic protein to a cell membrane.

[0007] An aspect of the present disclosure concerns a method of measuring the influence of a carrier peptide on the binding of a therapeutic protein to a cell membrane comprising: providing cells in a cell culture wherein the cell culture is maintained in a container; adding a composition comprising a labeled-therapeutic protein and a peptide carrier to the cell culture; incubating the cells culture with the composition to allow for binding of the peptide and the labeled-therapeutic protein to the cell cells in the cell culture; washing excess labeled-therapeutic1SUBSTITUTE SHEET (RULE 26)protein and excess peptide carrier from the container; and measuring the amount of labeled- therapeutic protein bound to the cells in the cell culture.

[0008] Another aspsect of the present disclores concerns a method of measuring the influence of a carrier peptide on the binding of a therapeutic protein to a cell membrane comprising: providing cells in a cell culture wherein the cell culture is maintained in a container; adding a peptide carrier to the cell culture; incubating the cell culture with the peptide carrier in the container; adding a labeled-therapeutic protein to the cell culture; incubating the cell culture with the labeled-therapeutic protein to allow for binding of the labeled-therapeutic protein to the cells in the cell culture; washing excess labeled-therapeutic protein and excess peptide carrier from the container; and measuring the amount of labeled-therapeutic protein bound to the cells in the cell culture.

[0009] Other aspect includes carrier peptides, botulinum toxin compositions comprising the same, and uses thereof.DESCRIPTION OF THE FIGURES

[0010] FIG. 1: Representative plot showing the impact of type of peptide backbone on binding of BoNT / A to a cell membrane.

[0011] FIG. 2: Representative plot showing the impact of the type and placement of a cell penetrating peptide on the amount of binding of BoNT / A to a cell membrane.

[0012] FIG. 3: Representative plot showing the impact of peptide backbone length on binding of BoNT / A to a cell membrane.DETAILED DESCRIPTION OF THE INVENTION

[0013] This present disclosure relates to methods for determining influence of carrier peptide on the binding of a therapeutic protein to a cell membrane.

[0014] In embodiments, a method of measuring the influence of a carrier peptide on the binding of a therapeutic protein to a cell membrane comprises: providing cells in a cell culture wherein the cell culture is maintained in a container, such as a well; adding a peptide carrier to the cell culture; incubating the cell culture with the peptide carrier in the container and optionally, removing excess peptide from the cell culture after incubation with the peptide; adding a labeled- therapeutic protein to the cell culture; incubating the cell culture with the labeled-therapeuticprotein to allow for binding of the labeled-therapeutic protein to the cells in the cell culture;washing excess labeled-therapeutic protein from the cell culture; and measuring the amount of labeled-therapeutic protein bound to the cells in the cell culture.

[0015] In another embodiment, a method of measuring the influence of a carrier peptide on the binding of a therapeutic protein to a cell membrane comprises: providing cells in a cell culture wherein the cell culture is maintained in a container; adding a composition comprising a labeled-therapeutic protein and a peptide carrier to the cell culture; incubating the cells culture with the composition to allow for binding of the peptide and the labeled-therapeutic protein to the cells in the cell culture; washing excess labeled-therapeutic protein from the cells; measuring the amount of labeled-therapeutic protein bound to the cells in the cell culture.

[0016] The therapeutic protein can be any protein, particulary one intended to besubcutaneously injected or topically (including mucosally) applied. In embodiments, the therapeutic protine is botulinum toxin or botulinum toxin Type A complex or 150 kd Type A botulinum toxin. Other types of botulinum toxin are also contemplated include Type B, C, D, E, F, G, and H, either complexed or the core neurotoxin thereof.

[0017] The label can be any compound that can be conjugated to protein and be detected with spectroscopy, such as UV / Vis, IR, or fluorescence. In embodiments, the label is a peroxidase or a horseradish peroxidase. In the case of peroxides, UV / VIS absorbance is used to measure the amount of labeled protein is in the container.

[0018] The method can comprise washing excess labeled-therapeutic protein from the container. A washing step can comprise flushing the container 1, 2, 3, 4, 5, 6, 7, or 8 time with 0.5 to 2% v / v or about 1 % v / v of non-ionic surfactant (such as polysorbate, such as polysorbate 20 or 80) in phosphate buffered saline (PCST is a common solution with the combination that can buse used to wash.)

[0019] Cells of the cell culture can be any cell suitable to culture and that is the target cell of a therapy or has properties similar to such cell. In embodiments, the cells of the cell culture can be neuronal cells, such as neuroblastoma cells. In embodiments, the cells are from a cell line selected from SiMa, Neuro-2a, NT2N, GT1-7, GNU, ND7 / 23, SH-Sy5Y, PC12, and HT-22.

[0020] A peptide carrier is configured to non-covalently bind to the therapeutic protein. Depending on the nature of the therapeutic protein, the peptide carrier can be a positively charged or negatively charged peptide carrier.

[0021] Generally, the positively-charged carrier (also referred to as a “positively charged backbone”) is typically a chain of atoms, either with groups in the chain carrying a positive charge at physiological pH, or with groups carrying a positive charge attached to side chains extending from the backbone. In certain preferred embodiments, the positively charged backbone is a cationic peptide. As used herein, the term “peptide” refers to an amino acid sequence, but carries no connotation with respect to the number of amino acid residues within the amino acid sequence. Accordingly, the term “peptide” may also encompass polypeptides and proteins. In certain preferred embodiments, the positively charged backbone itself will not have a defined enzymatic or therapeutic biologic activity. In certain embodiments, the backbone is a linear hydrocarbon backbone which is, in some embodiments, interrupted by heteroatoms selected from nitrogen, oxygen, sulfur, silicon and phosphorus. The majority of backbone chain atoms are usually carbon. Additionally, the backbone will often be a polymer of repeating units (e.g., amino acids, poly(ethyleneoxy), poly (propyleneamine), polyalkyleneimine, and the like) but can be a heteropolymer. In one group of embodiments, the positively charged backbone is a polypropyleneamine wherein a number of the amine nitrogen atoms are present as ammonium groups (tetra-substituted) carrying a positive charge. In another embodiment, the positively charged backbone is a nonpeptidyl polymer, which may be a hetero- or homo-polymer such as a polyalkyleneimine, for example a polyethyleneimine or polypropyleneimine, having a molecular weight of from about 10,000 to about 2,500,000, preferably from about 100,000 to about 1,800,000, and most preferably from about 500,000 to about 1,400,000. In another group of embodiments, the backbone has attached a plurality of side-chain moieties that include positively charged groups (e.g., ammonium groups, pyridinium groups, phosphonium groups, sulfonium groups, guanidinium groups, or amidinium groups). The sidechain moieties in this group of embodiments can be placed at spacings along the backbone that are consistent in separations or variable. Additionally, the length of the sidechains can be similar or dissimilar. For example, in one group of embodiments, the sidechains can be linear or branched hydrocarbon chains having from one to twenty carbon atoms and terminating at the distal end (away from the backbone) in one of the above-noted positively charged groups. The association between the positively chargedcarrier and the botulinum toxin is by non-covalent interaction, non-limiting examples of which include ionic interactions, hydrogen bonding, van der Waals forces, or combinations thereof. Examples of postively charged backbones and efficiency groups for use of the invention are described in U.S. Patent Nos. 8,623,811 and 9,211,248, which are hereby incorporated by reference in their entirety.

[0022] In one group of embodiments, the positively charged backbone is a polypeptide having multiple positively charged sidechain groups (e.g., lysine, arginine, ornithine, homoarginine, and the like). Preferably, the polypeptide has a molecular weight from about 100 to about 1,500,000, more preferably from about 500 to about 1,200,000, most preferably from about 1000 to about 1,000,000. One of skill in the art will appreciate that when amino acids are used in this portion of the invention, the sidechains can have either the D- or L-form (R or S configuration) at the center of attachment. In certain preferred embodiments, the polypeptide has a molecular weight from about 500 to about 5000, more preferably from 1000 to about 4000, more preferably from 2000 to about 3000. In other preferred embodiments, the polypeptide comprises 10 to 20 amino acids, or 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids, preferably polylysine.

[0023] Alternatively, the backbone may comprise amino acid analogs and / or synthetic amino acids. The backbone may also be an analog of a polypeptide such as a peptoid. See, for example, Kessler, Angew. Chem. Int. Ed. Engl. 32:543 (1993); Zuckermann et al. Chemtracts- Macromol. Chem. 4:80 (1992); and Simon et al. Proc. Nat'l. Acad. Sci. USA 89:9367 (1992)). Briefly, a peptoid is a polyglycine in which the sidechain is attached to the backbone nitrogen atoms rather than the oc-carbon atoms. As above, a portion of the sidechains will typically terminate in a positively charged group to provide a positively charged backbone component. Synthesis of peptoids is described in, for example, U.S. Patent No. 5,877,278, which is hereby incorporated by reference in its entirety. As the term is used herein, positively charged backbones that have a peptoid backbone construction are considered “non-peptide” as they are not composed of amino acids having naturally occurring sidechains at the alpha-carbon locations.

[0024] A variety of other backbones can be used employing, for example, steric or electronic mimics of polypeptides wherein the amide linkages of the peptide are replaced with surrogates such as ester linkages, thioamides (— CSNH-), reversed thioamide (— NHCS— ),aminomethylene (— NHCH2— ) or the reversed methyleneamino (— CH2NH— ) groups, ketomethylene (— COCH2— ) groups, phosphinate (— PO2RCH2--), phosphonamidate and phosphonamidate ester (— PO2RNH— ), reverse peptide (— NHCO— ), trans-alkene (— CR=CH— ), fluoroalkene (— CF=CH— ), dimethylene (--CH2CH2— ), thioether (— CH2S— ), hydroxyethylene (— CH(0H)CH2— ), methyleneoxy (— CH2O— ), tetrazole (CN4), sulfonamide (— SO2NH--), methylenesulfonamido (— CHRSO2NH— ), reversed sulfonamide (-- NHSO2— ), and backbones with malonate and / or gem-diamino-alkyl subunits, for example, as reviewed by Fletcher et al. ((1998) Chem. Rev. 98:763) and detailed by references cited therein. Many of the foregoing substitutions result in approximately isosteric polymer backbones relative to backbones formed from a-amino acids.

[0025] In each of the backbones provided above, sidechain groups can be appended that carry a positively charged group. For example, the sulfonamide-linked backbones (— SO2NH— and — NHSO2— ) can have sidechain groups attached to the nitrogen atoms. Similarly, the hydroxy ethylene (— CH(OH)CH2— ) linkage can bear a sidechain group attached to the hydroxy substituent. One of skill in the art can readily adapt the other linkage chemistries to provide positively charged sidechain groups using standard synthetic methods.

[0026] In one embodiment, the positively charged backbone is a polypeptide having protein transduction domains (also referred to as efficiency groups). As used herein, an efficiency group or protein transduction domain is any agent that has the effect of promoting the translocation of the positively charged backbone through a tissue or cell membrane. Non-limiting examples of protein transduction domains or efficiency groups include -(gly)ni-(arg)n2 (SEQ ID NO: 5), HIV- TAT or fragments thereof, or the protein transduction domain (PTD) of Antennapedia, or a fragment thereof, in which the subscript nl is an integer of from 0 to 20, more preferably 0 to 8, still more preferably 2 to 5, and the subscript n2 is independently an odd integer of from about 5 to about 25, more preferably about 7 to about 17, most preferably about 7 to about 13. In some embodiments, the HIV-TAT fragment does not contain the cysteine-rich region of the HIV-TAT molecule, in order to minimize the problems associated with disulfide aggregation. Preferably, the fragments of the HIV-TAT and Antennapedia protein transduction domains retain the protein transduction activity of the full protein. Still further preferred are those embodiments in which the HIV-TAT fragment has the amino acid sequence (gly)p-RGRDDRRQRRR-(gly)q(SEQ ID NO: 1),(gly)P-YGRKKRRQRRR-(gly)q(SEQ ID NO: 2) or (gly)P-RKKRRQRRR-(gly)q(SEQ ID NO: 3) wherein the subscripts p and q are each independently an integer of from 0 to 20, or wherein p and q are each independently the integer 1. In another embodiment, the fragment or efficiency group is attached to the backbone via either the C-terminus or the N-terminus of the fragment or amino acid sequence of the efficiency group. In certain preferred embodiments, p is one and q is zero or p is zero and q is one. Preferred HIV-TAT fragments are those in which the subscripts p and q are each independently integers of from 0 to 8, more preferably 0 to 5. In another preferred embodiment the positively charged side chain or branching group is the Antennapedia (Antp) protein transduction domain (PTD), or a fragment thereof that retains activity. These are known in the art, for instance, from Console et al., J. Biol. Chem. 278:35109 (2003) and a non-limiting example of an Antennapedia PTD contemplated by this invention is the PTD having the amino acid sequence SGRQIKTWFQNRRMKWKKC (SEQ ID NO: 6). In other embodiments, the positively charged carrier is a positively charged peptide having the amino acid sequence RKKRRQRRR-G-(K)15-G-RKKRRQRRR (SEQ ID NO: 4); or a positively charged peptide having the amino acid sequence YGRKKRRQR R-G-(K)I5-G-YGRKKRRQRRR (SEQ ID NO: 7); or a positively charged peptide having the amino acid sequences RGRDDRRQRRR-G-(K)is- G-RGRDDRRQRRR (SEQ ID NO: 8) for use in the compositions and methods of the invention.

[0027] Preferably the positively charged carrier includes side-chain positively charged protein transduction domains or positively charged efficiency groups in an amount of at least about 0.01%, as a percentage of the total carrier weight, preferably from about 0.01 to about 50 weight percent, more preferably from about 0.05 to about 45 weight percent, and most preferably from about 0.1 to about 30 weight %. For positively charged protein transduction domains having the formula -(gly)ni-(arg)n2 (SEQ ID NO: 5), a preferred range is from about 0.1 to about 25%.

[0028] In another embodiment, the backbone portion is a polylysine and positively charged protein transduction domains are attached to the lysine sidechain amino groups or to the C- or N- termini. In some preferred embodiments, the polylysine may have a molecular weight that is at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, or 6000 D, and less than about 2,000,000, 1,000,000, 500,000, 250,000, 100,000, 75,000, 50,000, and 25,000 D. Within the range of 100 to 2,000,000 D, it is contemplated that the lower and / or upper range may be increased or decreased, respectively, by 100, with each resulting sub-range being a specifically contemplated embodiment of the invention. In some exemplaryembodiments, the polylysine has a molecular weight from about 1,000 to about 1,500,000 D, from about 2,000 to about 800,000 D, or from about 3,000 to about 200,000 D. In other exemplary embodiments, the polylysine has molecular weight from about 100 to about 10,000 D, from about 500 to about 5,000 D, from about 1,000 to about 4,000 D, from about 1,500 to about 3,500 D or from about 2,000 to about 3,000 D. Preferred is a polylysine polypeptide having 10 to 20 lysines (SEQ ID NO: 9), more preferably, 15 lysines. In some embodiments, the polylysine contemplated by this invention can be any of the commercially available (Sigma Chemical Company, St. Louis, Mo., USA) polylysines such as, for example, polylysine having MW>70,000, polylysine having MW of 70,000 to 150,000, polylysine having MW 150,000 to 300,000 and polylysine having MW>300,000. The selection of an appropriate polylysine will depend on the remaining components of the composition and will be sufficient to provide an overall net positive charge to the composition and provide a length that is preferably from one to four times the combined length of the negatively charged components. Preferred positively charged protein transduction domains or efficiency groups include, for example, -gly-gly-gly-arg-arg-arg-arg-arg-arg-arg (-Gly^Arg? (SEQ ID NO: 10)) or HIV-TAT.

[0029] In another preferred embodiment the positively charged backbone is a polyalkyleneimine, non-limiting examples of which include polyethyleneimine, polypropyleneimine, and polybutyleneimine. In certain embodiments, the polyalkyleneimine has a molecular weight of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, or 6000 D, and less than about 2,000,000, 1,000,000, 500,000, 250,000, 100,000, 75,000, 50,000, and 25,000 D. Within the range of 100 to 2,000,000 D, it is contemplated that the lower and / or upper range may be increased or decreased, respectively, by 100, with each resulting sub-range being a specifically contemplated embodiment of the invention.

[0030] In other embodiments of this invention, the carrier is a relatively short polylysine or polyethyleneimine (PEI) backbone (which may be linear or branched) and which has positively charged branching groups. Without wishing to be constrained by theory, it is believed that such carriers are useful for minimizing uncontrolled aggregation of the backbones and botulinum toxin in a therapeutic composition, which causes the transport efficiency to decrease dramatically. When the carrier is a relatively short linear polylysine or PEI backbone, the backbone will have a molecular weight of less than 75,000 D, more preferably less than 30,000 D, and most preferably,less than 25,000 D. When the carrier is a relatively short branched polylysine or PEI backbone, however, the backbone will have a molecular weight less than 60,000 D, more preferably less than 55,000 D, and most preferably less than 50,000 D.

[0031] Postively charged carriers are listed in Table 1 of the Examples. Pharmaceutical composition that comprise a carrier peptide are also contemplated. Such embodiments comprise any carrier peptide of Table 1 and botulinum toxin, suh as botulinum toxin Type A or 150 kd Type A botulinum toxin. Such composition but with other types of toxin is also contemplated, with or without its native accessory proteins. In further embodiments, a method of reducing the appearance of fine lines or wrinkes or of treating migraine, cervical dystonia, or upper limb spasticity comprising administering an effective amount of such pharmaceutical compositions.EXAMPLESExample 1 - Cell-Based Carrier Peptide Enhancement Assay

[0032] To determine the influence of a carrier peptide on the binding of BoNT / A to a cell, a cellbased assay was performed. SiMa cells pre-coated with one of the peptides of Table 1 were plated in 96-well CellBind treated black, clear bottom plates at 7.5xl04cells / well in growth medium (RPMI-1640 + 10% FBS) and allowed to attach for two days. Horseradish peroxidase (HRP) was conjugated to 150 kDa BoNT / A using the Mix-n-Stain HRP Antibody labeling kit (Biotium, Fremont CA, USA) according to the manufacturer’s protocol. The cells were then incubated with a fixed amount of HRP -labeled 150 kDa BoNT / A (100 pL at 3.3 nM) for 1 hour at 37 °C. Then the plate was washed again five times in PBST followed by the addition of 100 pL of TMB substrate for 10-15 minutes to allow color to develop. The reaction was stopped by the addition of 50 pL of a 1 M sulfuric acid solution and absorbance was read at 450 nm with an M5 Spectramax plate reader.

[0033] Table 1binding of BoNT / A between PTD-K15-PTD and PTD-R15-PTD, and the results are shown in FIG.1. The impact of different cell penetrating peptides (PTD vs. PTD4) and the number of cell penetrating peptides on a polylysine backbone is studied and the results are shown in FIG. 2. The effect of the backbone length was studied by comparing the impact of PTD-K5-PTD and PTD- K10-PTD and PTD-K15-PTD, and the results are shown in FIG. 3. This study demonstrated that RTP004 had the highest percent change in binding when compared to no peptide present in all of these studies.

Claims

CLAIMSI / We claim:

1. A method of measuring the influence of a carrier peptide on the binding of a therapeutic protein to a cell membrane comprising: providing a cell culture comprising cells, wherein the cell culture is maintained in a container; adding a peptide carrier to the cell culture; incubating the cell culture with the peptide carrier in the container ; adding a labeled-therapeutic protein to the cell culture; incubating the cell culture with the labeled-therapeutic protein to allow for binding of the labeled-therapeutic protein to the cells in the cell culture; washing excess labeled-therapeutic protein and excess peptide carrier from the cell culture; and measuring the amount of labeled-therapeutic protein bound to the cells in the cell culture. A method of measuring the influence of a carrier peptide on the binding of a therapeutic protein to a cell membrane comprising: providing a cell culture comprising cells, wherein the cell culture is maintained in a container; adding a composition comprising a labeled-therapeutic protein and a peptide carrier to the cell culture; incubating the cells culture with the composition to allow for binding of the peptide and the labeled-therapeutic protein to the cells in the cell culture; washing excess labeled-therapeutic protein and excess peptide carrier from the cells; measuring the amount of labeled-therapeutic protein bound to the cells in the cell culture.3 The method of claim 1 or 2, wherein therapeutic protein of the labeled-therapeutic protein is botulinum toxin or botulinum toxin Type A complex or 150 kd Type A botulinum toxin. The method of any one of the foregoing claims, wherein the label of the labeled therapeutic protein is a peroxidase or a horseradish peroxidase.5 The method of any one of the foregoing claims, wherein measuring the amount of labeled- therapeutic protein comprises measuring an amount of UV / VIS absorbance of the label in the cell culture.

6. The method of any one of the foregoing claims, wherein washing excess labeled-therapeutic protein from the well comprising flushing the well 1, 2, 3, 4, 5, 6, 7, or 8 time with 0.5 to 2% v / v or about 1 % v / v of non-ionic surfactant in phosphate buffered saline or with PBST.

7. The method of any one of the foregoing claims, wherein the cell culture comprises neuroblastoma cells or neuroblastoma SiMa cells or neuroblastoma N2a cells.8 The method of any one of the foregoing claims, wherein the peptide carrier is positively charged.9 The method of claim 8, wherein the peptide carrier comprises polylysine.10 The method of claim 8 or 9, wherein the peptide carrier comprises one or more cell penetrating peptides.11 The method of any one of claims 1 to 7 or 10, wherein the peptide carrier is negatively charged.12 The method of claim 1, comprising removing excess peptide from the cell culture after incubation with the peptide.13 A carrier peptide according to any one of SEQ ID Nos: 11 to 18.14 A pharmaceutical composition comparising a carrier peptide according to claim 13 and botulinum toxin.15 The composition of claim 14, wherein the botulinum toxin is botulinum toxin Type A complex or 150 kd Type A botulinum toxin.16 A method of reducing the appearance of fine lines or wrinkes or of treating migraine, cervical dystonia, or upper limb spasticity comprising administering an effective amount of a pharmaceutical composition according to claims 14 or 15.17 The method of any one of claims 1 to 12, whrerein the cells are neuronal cells, such as cells of a cell line selected from SiMa, Neuro-2a, NT2N, GT1-7, GNU, ND7 / 23, SH-Sy5Y, PC12, and HT-22.

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