Methods for quantifying frizzled and frizzled fusion proteins

Abstract

The present disclosure provides methods of determining the amount of FXN or FXN fusion protein in a sample (e.g., a tissue sample) by using mass spectrometry.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 880068, filed July 29, 2019, and U.S. Provisional Patent Application No. 62 / 936293, filed November 15, 2019. The entire contents of each of the preceding applications are incorporated herein by reference.

[0003] sequence list

[0004] This application contains a sequence list electronically submitted in ASCII format, the entire contents of which are incorporated herein by reference. The ASCII copy created on July 28, 2020, is named 130197-00920_SL.txt and is 9786 bytes in size. Background Technology

[0005] Friedreich ataxia (FRDA) is a rare, inherited, progressive neurodegenerative disorder caused by mutations in the gene encoding the ataxia protein (FXN). FXN is an essential and systemically conserved protein found in cells throughout the body, with the highest levels in the heart, spinal cord, liver, pancreas, and skeletal muscle. FXN is encoded in the cell nucleus, expressed in the cytoplasm, and introduced into the mitochondria, where it is processed into its mature form. In humans, the full-length 210-amino acid hFXN (hFXN) is... 1-210 The protein (23.1 kDa) contains a typical mitochondrial targeting sequence (MTS) at its N-terminus. When introduced into the mitochondrial matrix, it is processed in a two-step cleavage by matrix mitochondrial processing peptidase (MPP). The resulting protein is a mature hFXN protein (hFXN) of 130 amino acids and 14.2 kDa. 81-210 ).

[0006] TAT-GG-FXN is an investigational 24.9 kDa fusion protein as an FXN replacement therapy to restore FXN functional levels in the mitochondria of FRDA patients. TAT-GG-FXN comprises an HIV-TAT peptide linked to the N-terminus of the full-length hFXN protein. The mechanism of action of TAT-GG-FXN relies on the cell-penetrating ability of the HIV-TAT peptide to deliver TAT-GG-FXN into cells, where it is subsequently processed into mature hFXN after translocation to the mitochondria. TAT-GG-FXN (SEQ ID NO: 11) is described in U.S. Provisional Patent Application 62 / 891029, the entire contents of which are incorporated herein by reference and are as follows.

[0007] To advance the development of FXN replacement therapies, new and reliable methods are needed to accurately and precisely measure the levels of FXN and FXN fusion proteins in various biological fluids and tissues of subjects. Summary of the Invention

[0008] In one aspect, this disclosure provides a method for determining the amount of unprocessed fusion protein (FXN) in a sample, the method comprising determining the amount of peptide in the sample by using mass spectrometry (MS).

[0009] The peptide comprises or consists of the amino acid sequence GGMWTLGR (SEQ ID NO: 12); and

[0010] The unprocessed FXN fusion protein comprises or consists of the following from the N-terminus to the C-terminus: a cell-penetrating peptide (CPP); a mitochondrial targeting sequence (MTS) comprising or consisting of SEQ ID NO: 3; and FXN comprising or consisting of SEQ ID NO: 2.

[0011] In one embodiment, the sample was obtained from a subject who was administered unprocessed FXN fusion protein.

[0012] In one embodiment, the CPP comprises or consists of HIV-TAT (SEQ ID NO: 4) or HIV-TAT+M (SEQ ID NO: 15).

[0013] In one embodiment, the unprocessed FXN fusion protein is TAT-GG-FXN (SEQ ID NO: 11).

[0014] In one embodiment, the method includes purifying the unprocessed FXN fusion protein from the sample by immune capture prior to MS.

[0015] In one implementation, the method includes the following steps:

[0016] a) Purify the unprocessed FXN fusion protein from the sample by immunocapture to obtain an immunocapture complex containing the unprocessed FXN fusion protein;

[0017] b) Digesting the immune-capture complex containing the unprocessed FXN fusion protein with trypsin to produce a peptide containing or consisting of SEQ ID NO: 12; and

[0018] c) The amount of peptide containing or composed of SEQ ID NO: 12 was determined by liquid chromatography and tandem mass spectrometry (LC / MS-MS).

[0019] In one embodiment, in step c), liquid chromatography (LC) includes reversed-phase chromatography.

[0020] In one implementation, tandem mass spectrometry (MS-MS) includes monitoring the transition 439.2→632.3.

[0021] In one implementation, tandem mass spectrometry (MS-MS) includes monitoring the transition 439.2→446.3.

[0022] In one implementation, an antigen-binding domain comprising a specific binding domain to the FXN fusion protein is used for immune capture.

[0023] In one embodiment, the binding protein specifically binds to FXN present in the FXN fusion protein.

[0024] In one embodiment, the binding protein specifically binds to CPP present in the FXN fusion protein.

[0025] In one embodiment, the FXN fusion protein comprises HIV-TAT (SEQ ID NO: 4) or HIV-TAT+M (SEQ ID NO: 15), wherein the binding protein specifically binds to HIV-TAT.

[0026] In one implementation, the sample is a tissue sample.

[0027] In one implementation, the tissue sample comprises an oral swab.

[0028] In one implementation, the tissue sample includes a skin biopsy.

[0029] In one implementation, the sample is derived from the subject's blood.

[0030] In one implementation, the sample contains platelets.

[0031] In one implementation, the sample is a plasma sample.

[0032] In one implementation, the method has an accuracy of about 20% or less, as measured by the % coefficient of variation (%CV).

[0033] In one embodiment, the method has an accuracy of about 15% or less, about 14% or less, about 13% or less, about 12% or less, about 11% or less, about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, or about 1% or less, as measured by %CV.

[0034] In one implementation, the method has an accuracy of about 80% to about 120%.

[0035] In one embodiment, the method is linear for concentrations of unprocessed FXN fusion protein ranging from about 0.250 ng / mL to about 25.000 ng / mL.

[0036] On the other hand, this disclosure provides a method for monitoring the processing of unprocessed FXN fusion proteins in a sample, the method comprising:

[0037] Obtain samples from the subject at at least two time intervals; and

[0038] The amount of unprocessed FXN fusion protein in the time-separated samples was determined using the method of any one of claims 1-23.

[0039] In one implementation, the sample was derived from a subject who was given unprocessed FXN fusion protein.

[0040] In another aspect, this disclosure provides a method for adjusting a dosing regimen for administering unprocessed FXN fusion protein, the method comprising:

[0041] Obtain samples from subjects who were administered the unprocessed FXN fusion protein at at least two separate time points;

[0042] The amount of unprocessed FXN fusion protein in the time-separated samples was determined using the method of any one of claims 1-23; and

[0043] Based on the time-varying amount of unprocessed FXN fusion protein observed in subjects, the dose of unprocessed FXN fusion protein administered to subjects and / or the frequency of administration of unprocessed FXN fusion protein to subjects were adjusted.

[0044] In another aspect, this disclosure provides a method for determining the amount of unprocessed FXN fusion protein, such as TAT-GG-FXN (SEQ ID NO: 11), in a sample, the method comprising determining the amount of at least one peptide in the sample by using MS.

[0045] The at least one peptide is selected from the group consisting of:

[0046] a) A peptide comprising the amino acid sequence SGTLGHPGSLDETTYER (SEQ ID NO: 13) or thereof; and

[0047] b) Contains the amino acid sequence LGGDLGTYVINK (SEQ ID NO: 14) or a peptide thereof;

[0048] The method described therein includes purifying unprocessed TAT-GG-FXN from the sample by immunocapture prior to MS;

[0049] The immune capture is performed using a binding protein containing an antigen-binding domain, wherein the binding protein is capable of binding the TAT protein transduction domain.

[0050] In one embodiment, the sample was obtained from a subject who was administered unprocessed TAT-GG-FXN (SEQ NO: 11).

[0051] In one implementation, the method includes the following steps:

[0052] a) Purify unprocessed FXN fusion protein, such as TAT-GG-FXN, from the sample by immunocapture to obtain an immunocapture complex containing unprocessed FXN fusion protein, such as TAT-GG-FXN.

[0053] b) Digesting the immune-captured complex containing an unprocessed FXN fusion protein, such as TAT-GG-FXN, with a protease, such as trypsin, to produce a peptide containing or comprising SEQ ID NO: 13 and / or a peptide containing or comprising SEQ ID NO: 14; and

[0054] c) The amount of peptides containing or consisting of SEQ ID NO: 13 and / or peptides containing or consisting of SEQ ID NO: 14 was determined by LC / MS-MS.

[0055] In one embodiment, in step c), LC comprises reversed-phase chromatography.

[0056] In one embodiment, MS-MS includes monitoring transitions 607.3→669.3 of peptides comprising or consisting of SEQ ID NO: 13 and SEQ ID NO: 14 ...

[0057] In one implementation, the sample is a tissue sample.

[0058] In one implementation, the tissue sample comprises an oral swab.

[0059] In one implementation, the tissue sample includes a skin biopsy.

[0060] In one implementation, the sample is derived from the subject's blood.

[0061] In one implementation, the sample contains platelets.

[0062] In one implementation, the sample is a plasma sample.

[0063] In one implementation, the method has an accuracy of about 20% or less, as measured by the % coefficient of variation (%CV).

[0064] In one implementation, the method has an accuracy of about 80% to about 120%.

[0065] In one embodiment, the method is linear for concentrations of unprocessed TAT-GG-FXN ranging from 0.250 ng / mL to 25.000 ng / mL.

[0066] In another aspect, this disclosure provides a method for determining the amount of FXN in a tissue sample, the method comprising determining the amount of at least one peptide of the FXN from the sample by means of MS.

[0067] The at least one peptide is selected from the group consisting of:

[0068] a) A peptide comprising the amino acid sequence SGTLGHPGSLDETTYER (SEQ ID NO: 13) or thereof; and

[0069] b) Contains the amino acid sequence LGGDLGTYVINK (SEQ ID NO: 14) or a peptide thereof;

[0070] The method is characterized by at least one of the following:

[0071] This method has an accuracy of 20% or less, as measured by the % coefficient of variation (%CV); and

[0072] This method has an accuracy of approximately 80% to approximately 120%.

[0073] In one implementation, the sample is obtained from the subject.

[0074] In one implementation, the subject was administered unprocessed FXN fusion protein.

[0075] In one embodiment, the unprocessed FXN fusion protein comprises or consists of the following from the N-terminus to the C-terminus: a cell-penetrating peptide (CPP), a mitochondrial targeting sequence (MTS) of SEQ ID NO: 3, and FXN.

[0076] In one embodiment, the CPP comprises or consists of HIV-TAT (SEQ ID NO: 4) or HIV-TAT+M (SEQ ID NO: 15).

[0077] In one embodiment, the unprocessed FXN fusion protein is TAT-GG-FXN (SEQ ID NO: 11).

[0078] In one embodiment, the method includes purifying FXN from the sample by immune capture prior to MS.

[0079] In one implementation, the method includes the following steps:

[0080] a) Purify FXN from the sample by immunocapture to obtain an immunocapture complex containing FXN;

[0081] b) Digesting the FXN-containing immune capture complex with a protease such as trypsin to produce a peptide comprising or consisting of SEQ ID NO: 13 and / or a peptide comprising or consisting of SEQ ID NO: 14; and

[0082] c) The amount of at least one peptide selected from peptides comprising or consisting of SEQ ID NO: 13 and / or peptides comprising or consisting of SEQ ID NO: 14 was determined by LC / MS-MS.

[0083] In one embodiment, in step c), LC comprises reversed-phase chromatography.

[0084] In one embodiment, tandem mass spectrometry (MS-MS) includes monitoring transitions 607.3→669.3 of peptides containing or composed of SEQ ID NO: 13 and SEQ ID NO: 14, or SEQ ID NO: 14, and transitions 625.3→794.4 of peptides containing or composed of SEQ ID NO: 14.

[0085] In one implementation, an immune capture is performed using a binding protein containing an antigen-binding domain that specifically binds to FXN.

[0086] In one embodiment, the FXN is part of an FXN fusion protein that also contains HIV-TAT (SEQ ID NO: 4) or HIV-TAT+M (SEQ ID NO: 15), wherein the binding protein specifically binds to HIV-TAT.

[0087] In one implementation, the tissue sample comprises an oral swab.

[0088] In one implementation, the tissue sample includes a skin biopsy.

[0089] In another aspect, this disclosure provides a method for determining the amount of FXN in a skin sample, the method comprising determining the amount of at least one peptide of the FXN from the skin sample by using MS.

[0090] The at least one peptide is selected from the group consisting of:

[0091] a) Contains the amino acid sequence GGMWTLGR (SEQ ID NO: 12) or a peptide thereof;

[0092] b) A peptide containing the amino acid sequence SGTLGHPGSLDETTYER (SEQ ID NO: 13) or composed thereof; and

[0093] c) Contains the amino acid sequence LGGDLGTYVINK (SEQ ID NO: 14) or a peptide thereof.

[0094] In one implementation, the skin sample is obtained from the subject.

[0095] In one implementation, the subject was administered unprocessed FXN fusion protein.

[0096] In one embodiment, the unprocessed FXN fusion protein comprises or consists of the following from the N-terminus to the C-terminus: a cell-penetrating peptide (CPP), a mitochondrial targeting sequence (MTS) of SEQ ID NO: 3, and FXN, such as the FXN of SEQ ID NO: 2.

[0097] In one embodiment, the CPP comprises or consists of HIV-TAT (SEQ ID NO: 4) or HIV-TAT+M (SEQ ID NO: 15).

[0098] In one embodiment, the unprocessed FXN fusion protein is TAT-GG-FXN (SEQ ID NO: 11).

[0099] In one embodiment, the method includes purifying FXN from the skin sample via immune capture prior to MS.

[0100] In one implementation, the method includes the following steps:

[0101] a) Purify FXN from the skin sample by immune capture to obtain an immune capture complex containing FXN;

[0102] b) Digesting the FXN-containing immune capture complex with a protease such as trypsin to produce a peptide comprising or consisting of SEQ ID NO: 12 and / or a peptide comprising or consisting of SEQ ID NO: 13 and / or a peptide comprising or consisting of SEQ ID NO: 14; and

[0103] c) The amount of at least one peptide selected from peptides comprising or consisting of SEQ ID NO: 12 and / or peptides comprising or consisting of SEQ ID NO: 13 and / or peptides comprising or consisting of SEQ ID NO: 14 was determined by LC / MS-MS.

[0104] In one embodiment, the immune capture is performed using a binding protein containing an antigen-binding domain that specifically binds to FXN.

[0105] In one embodiment, the FXN is part of an FXN fusion protein that also contains HIV-TAT (SEQ ID NO: 4) or HIV-TAT+M (SEQ ID NO: 15), wherein the binding protein specifically binds to HIV-TAT. Attached Figure Description

[0106] Figure 1 This is a description of a method for preparing biological samples for quantification of TAT-GG-FXN (SEQ ID NO: 11).

[0107] Figure 2 This is a diagram illustrating sample preparation for hybrid LC-MS / MS analysis. Figure 2 SEQ ID NO 12-14 are disclosed in the order of their appearance.

[0108] Figure 3 This is a series of graphs showing the concentration levels of TAT-GG-FXN (SEQ ID NO: 11) in monkey tissues (N=6) 14 days after repeated subcutaneous (SC) administration at 15 mg / kg BID. Data are expressed as pg TAT-GG-FXN / μg total protein.

[0109] Figure 4 These are a series of representative chromatograms of TAT-GG-FXN trypsin peptide in monkey tissues before administration (day 1) and 14 days after repeated administration of SC at 15 mg / kg BID. Detailed Implementation

[0110] This disclosure provides a method for determining the amount of ataxia protein (FXN) or FXN fusion protein in a sample using mass spectrometry (MS). FXN is a protein associated with the disease Friedreich ataxia (FRDA). FRDA is a hereditary, progressive neurodegenerative disorder caused by mutations in the gene encoding FXN. The method of this disclosure can be used, for example, to monitor the level of FXN in a subject, such as a subject receiving FXN as a protein replacement therapy for the treatment of FRDA. The method of the present invention can also be used to monitor the processing of FXN fusion proteins, such as TAT-GG-FXN (SEQ ID NO: 11), after adding the FXN fusion protein to an in vitro sample or administering it to a subject. The method of this disclosure can also be used to monitor the level of FXN or FXN fusion protein in a subject, such as a subject receiving the FXN fusion protein, and to adjust the dose or frequency of administration of the FXN fusion protein.

[0111] FXN and FXN fusion protein

[0112] FXN is an essential and systemogenically conserved protein found in cells throughout the body, with the highest levels in the heart, spinal cord, liver, pancreas, and skeletal muscle. FXN is encoded in the cell nucleus, expressed in the cytoplasm, and introduced into the mitochondria, where it is processed into its mature form. In humans, the full-length 210-amino acid hFXN (hFXN) is... N1-210 The protein (23.1 kDa) contains a typical mitochondrial targeting sequence (MTS) at its N-terminus. When introduced into the mitochondrial matrix, it is processed in a two-step cleavage by matrix mitochondrial processing peptidase (MPP). The resulting protein is a mature hFXN protein (hFXN) of 130 amino acids and 14.2 kDa. 81-210 The sequences of full-length hFXN and mature hFXN are shown in Table 1 below.

[0113] Table 1. Sequences of full-length hFXN and mature hFXN

[0114]

[0115]

[0116] The full-length hFXN (SEQ ID NO: 1) contains mature hFXN (SEQ ID NO: 2) and a mitochondrial targeting sequence (MTS) with the amino acid sequence MWTLRRAVAGLLASPSPAQAQTLTRVPRPAELAPLCGRRGLRTDIDATCTPRRASSNQRGLNQIWNVKKQSVYLMNLRK (SEQ ID NO: 3).

[0117] In some embodiments, the FXN measured by the methods of this disclosure may be a mammalian FXN. In some embodiments, the FXN measured by the methods of this disclosure may be a human FXN (hFXN). In some embodiments, the FXN measured by the methods of this invention may be a monkey FXN, such as the FXN from a cynomolgus monkey.

[0118] In some aspects, the FXN quantified by the methods provided in this disclosure may be endogenous FXN, i.e., naturally present in in vitro samples, such as biological samples from a subject or FXN in the subject. In other aspects, the FXN quantified by the methods provided in this disclosure may be exogenous FXN, i.e., FXN that has been added to an in vitro sample, or FXN that has been administered to a subject, for example, and therefore present in a biological sample from the subject. Exogenous FXN may be contained in FXN fusion proteins, such as TAT-GG-FXN (SEQ ID NO: 11).

[0119] As used herein, the term "FXN fusion protein" refers to an artificial polypeptide containing FXN (e.g., full-length hFXN (SEQ ID NO: 1) or mature hFXN (SEQ ID NO: 2)). In some embodiments, the FXN fusion protein also contains a cell-penetrating peptide (CPP).

[0120] As used herein, the term "cell-penetrating peptide" or "CPP" refers to a short peptide sequence, typically 5-30 amino acids in length, that facilitates cellular uptake of various molecular cargoes, such as proteins. In the context of this invention, CPPs present in the FXN fusion protein facilitate the delivery of the FXN fusion protein to cells, such as recipient cells.

[0121] CPPs can be polycationic, meaning they contain amino acid compositions with high relative abundance of positively charged amino acids such as lysine or arginine. CPPs can also be amphiphilic, meaning they have sequences containing alternating patterns of polar / charged amino acids and nonpolar, hydrophobic amino acids. CPPs can also be hydrophobic, meaning they contain only nonpolar residues with low net charge, or hydrophobic amino acid groups that are crucial for cellular uptake.

[0122] The CPP that can be included in the FXN fusion protein useful in the context of this invention can be any CPP known to those skilled in the art. For example, the CPP can be any CPP listed in the CPPsite2.0 database of cell-penetrating peptides, the entire contents of which are incorporated herein by reference. For example, a CPP useful in the context of this invention can be a cell-penetrating peptide derived from a protein selected from: HIV transactivator peptide (HIV-TAT), galanin, mast cell degranulation peptide, transportan, penetratin, polyarginine, or VP22. In some embodiments, the CPP contains the transduction domain of the TAT protein, comprising amino acids 47-57 of the full-length HIV-TAT protein of 86 amino acids (this 11-amino acid peptide may also be referred to herein as "HIV-TAT"; SEQ ID NO: 4). In one embodiment, the CPP consists of HIV-TAT (SEQ ID NO: 4). In some embodiments, the CPP comprises amino acids 47-57 of the full-length HIV-TAT protein, consisting of 86 amino acids, with a methionine added at the amino terminus for initiation (12 AAs; "HIV-TAT+M"): MYGRKKRRQRRR (SEQ ID NO: 15). Table 1 below lists the amino acid sequences of exemplary CPPs.

[0123] Table 1. Exemplary CPPs and Corresponding Sequences

[0124]

[0125] In some embodiments, the CPP contained in the FXN fusion protein is HIV-TAT (SEQ ID NO: 4) or HIV-TAT+M (SEQ ID NO: 15). In some embodiments, the FXN fusion protein comprises full-length FXN (e.g., SEQ ID NO: 1, and HIV-TAT, e.g., SEQ ID NO: 4 or HIV-TAT+M (SEQ ID NO: 15)) as the CPP.

[0126] In some embodiments, in the FXN fusion protein of this disclosure, the CPP can be fused to an FXN, such as a full-length FXN, via a linker to form a single polypeptide chain. In one embodiment, the linker may comprise the amino acid sequence GG. In one embodiment, the linker consists of the amino acid sequence GG. In some embodiments, the CPP is located at the N-terminus and the FXN is located at the C-terminus. In some embodiments, the CPP is located at the C-terminus and the FXN is located at the N-terminus. It should be understood that in some embodiments, the methods provided herein can be used to detect FXN fusion proteins comprising any CPP and FXN, such as a full-length FXN, wherein the CPP and FXN are fused via a GG linker.

[0127] In some embodiments, the FXN fusion protein comprises the following amino acid sequence (224 amino acids): MYGRKKRRQRRRGGMWTLGRRAVAGLLASPSPAQAQTLTRVPRPAELAPLCGRRGLRTDIDATCTPRRASSNQRGLNQIWNVKKQSVYLMNLRKSGTLGHPGSLDETTYERLAEETLDSLAEFFEDLADKPYTFEDYDVSFGSGVLTVKLGGDLGTYVINKQTPNKQIWLSSPSSGPKRYDWTGKNWVYSHDGVSLHELLAAELTKALKTKLDLSSLAYSGKDA (Sequence No.: 11). In some embodiments, the FXN fusion protein consists of the amino acid sequence of SEQ ID NO: 11, namely TAT-GG-FXN. TAT-GG-FXN is further described in U.S. Provisional Patent Application No. 62 / 891029, the entire contents of which are incorporated herein by reference and are as follows.

[0128] In some aspects, the CPP (e.g., HIV-TAT) present in the FXN fusion protein of this disclosure facilitates the delivery of the FXN fusion protein into cells, such as cells that may be present in vitro, outside the body, or in a subject. Once intracellular, the FXN fusion protein can be processed by cellular mechanisms to remove the CPP, such as HIV-TAT, from the FXN. Therefore, the term "unprocessed FXN fusion protein" as used herein refers to the intact FXN fusion protein, i.e., the full-length FXN fusion protein, or an FXN fusion protein from which the CPP (e.g., HIV-TAT) has not yet been removed. Such an FXN fusion protein may be an FXN fusion protein that has not yet been delivered into cells. For example, after administration of the FXN fusion protein to a subject, the unprocessed FXN fusion protein may be present in the subject's circulation, such as in plasma. In some embodiments, the FXN fusion protein may be an FXN fusion protein that has been delivered into cells but has not been processed by cellular mechanisms to remove the CPP, such as HIV-TAT. In some embodiments, the unprocessed FXN fusion protein comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, the unprocessed FXN fusion protein consists of the amino acid sequence of TAT-GG-FXN (SEQ ID NO: 11).

[0129] Mass spectrometry-based methods for quantifying FXN and FXN fusion proteins

[0130] This disclosure provides a mass spectrometry-based method for determining the amount of FXN or FXN fusion proteins, such as unprocessed FXN fusion proteins, like TAT-GG-FXN (SEQ ID NO: 11), in a sample. In some embodiments, the sample may be an in vitro sample. In some embodiments, the sample may be a biological sample, such as one containing biological fluids or tissues. In some embodiments, the biological sample (e.g., containing biological fluids or tissues) is derived from a subject, such as a subject with FRDA and / or a subject who has received unprocessed FXN fusion proteins.

[0131] In some respects, the amount of FXN or FXN fusion protein in a sample determined using the methods of this disclosure is an unnormalized amount. In other respects, the amount of FXN or FXN fusion protein determined using the methods of this disclosure is normalized, for example, normalized to the total amount of protein present in the sample. The total amount of protein present in the sample can be determined by any method known in the art for determining the amount of total protein, such as the BCA assay.

[0132] In some aspects, the methods of this disclosure are characterized by high precision and accuracy in measuring the amount of FXN or FXN fusion protein. For example, the methods provided by the present invention may be characterized by an accuracy of about 20% or less, such as about 15% or less, about 14% or less, about 13% or less, about 12% or less, about 11% or less, about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, or about 1% or less, as measured by % coefficient of variation (%CV). In some aspects, the methods provided by the present invention may be characterized by %CV of 10% or less.

[0133] In some aspects, the methods of this disclosure may be characterized by an accuracy of about 80% to about 120%, for example about 80% to about 110%, or about 80% to about 100%, or about 85% to about 120%, or about 85% to about 110%, or about 85% to about 100%, or about 90% to about 120%, or about 90% to about 110%, or about 90% to about 100%, or about 95% to about 120%, or about 95% to about 110%, or about 95% to about 100%.

[0134] In some respects, when used to measure the amount of FXN fusion protein, the methods of this disclosure can be linear for the concentration of unprocessed FXN fusion protein ranging from about 0.250 ng / mL to about 25.000 ng / mL.

[0135] In some respects, when used to measure the amount of TAT-GG-FXN (SEQ ID NO: 11), the methods of this disclosure can be linear for the concentration of unprocessed TAT-GG-FXN ranging from about 0.250 ng / mL to about 25.000 ng / mL.

[0136] In some embodiments, the method of this disclosure may include purifying an immunocapture complex containing FXN or FXN fusion protein present in an immunocapture sample; digesting the immunocapture complex with a protease to obtain peptides derived from FXN or FXN fusion protein; and analyzing the peptides derived from FXN or FXN fusion protein by liquid chromatography and tandem mass spectrometry (LC-MS / MS) to determine the amount of FXN or FXN fusion protein present in the sample. In some embodiments, the protease may be selected from trypsin, chymotrypsin, LysC, LysN, AspN, GluC, and ArgC. In one embodiment, the protease is trypsin.

[0137] In some aspects, the methods of this disclosure include detecting peptides derived from FXN or FXN fusion proteins, i.e., trypsin peptides derived from FXN and / or FXN fusion proteins, after digestion of the immunocapture complex with a protease such as trypsin. Trypsin peptides that can be monitored and / or quantified in the methods of this disclosure are summarized in Table 2 below.

[0138] Table 2. Peptides quantified and / or monitored in the method of the present invention

[0139]

[0140]

[0141] In some aspects, the methods of the present invention include detecting and / or quantifying at least one peptide listed in Table 2. In some aspects, the methods of the present invention include tandem mass spectrometry (MS / MS). For example, in some embodiments, MS / MS can be used to detect and / or quantify the linker peptide (peptide GGM) by monitoring the transition 439.2→632.3 and / or the transition 439.2→446.3. In some aspects, MS / MS can be used to detect and / or quantify the peptide SGT by monitoring the transition 607.3→669.3. In some aspects, MS / MS can be used to detect and / or quantify the peptide LGG by monitoring the transition 625.3→794.4.

[0142] In some aspects, the method of the present invention may involve performing liquid chromatography (LC) prior to mass spectrometry. For example, the LC may be reversed-phase LC.

[0143] In some embodiments, the method of the present invention may involve purifying the unprocessed FXN fusion protein from the sample by immune capture prior to MS. For example, the method of this disclosure may include the following steps:

[0144] a) Purify the unprocessed FXN fusion protein from the sample by immunocapture to obtain an immunocapture complex containing the unprocessed FXN fusion protein;

[0145] b) Digesting the immune-capture complex containing the unprocessed FXN fusion protein with a protease such as trypsin to produce, for example, a peptide containing SEQ ID NO: 12; and / or a peptide containing SEQ ID NO: 13; and / or a peptide containing SEQ ID NO: 14; and

[0146] c) The amount of peptide containing SEQ ID NO: 12; and / or peptide containing SEQ ID NO: 13; and / or peptide containing SEQ ID NO: 14 was determined by liquid chromatography and tandem mass spectrometry (LC / MS-MS).

[0147] In some embodiments, the peptides produced by the method include the peptide composed of SEQ ID NO: 12; and / or the peptide composed of SEQ ID NO: 13; and / or the peptide composed of SEQ ID NO: 14. In some embodiments, the amount of peptide determined by liquid chromatography and tandem mass spectrometry (LC / MS-MS) in the method includes the peptide composed of SEQ ID NO: 12; and / or the peptide composed of SEQ ID NO: 13; and / or the peptide composed of SEQ ID NO: 14.

[0148] In the context of this invention, immune capture can be performed using binding proteins, such as antibodies, that include an antigen-binding domain that specifically binds to FXN or FXN fusion proteins. In some embodiments, the binding protein, such as the antibody, includes an antigen-binding domain that specifically binds to the FXN portion of an FXN or FXN fusion protein.

[0149] In some embodiments, the binding protein, such as an antibody, includes an antigen-binding domain that specifically binds to an FXN fusion protein but not to FXN, such as endogenous FXN present in the sample. For example, in some embodiments, the binding protein, such as an antibody, includes an antigen-binding domain that specifically binds to CPP present in an FXN fusion protein such as HIV-TAT.

[0150] In some aspects, this disclosure provides a method for determining the amount of unprocessed FXN fusion protein in a sample, the method comprising determining the amount of peptides in the sample by mass spectrometry (MS), wherein the peptides comprise the amino acid sequence GGMWTLGR (SEQ ID NO: 12); and wherein the unprocessed FXN fusion protein comprises, from the N-terminus to the C-terminus: a cell-penetrating peptide (CPP); a mitochondrial targeting sequence (MTS) comprising SEQ ID NO: 3; and FXN comprising SEQ ID NO: 2.

[0151] In some embodiments, the CPP comprises HIV-TAT (SEQ ID NO: 4) or HIV-TAT+M (SEQ ID NO: 15). In some embodiments, the unprocessed FXN fusion protein comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, the unprocessed FXN fusion protein consists of the amino acid sequence TAT-GG-FXN (SEQ ID NO: 11).

[0152] In some embodiments, this disclosure provides a method for determining the amount of unprocessed TAT-GG-FXN (SEQ ID NO: 11) in a sample, the method comprising determining the amount of at least one peptide in the sample using MS, wherein the at least one peptide is selected from: a) a peptide comprising the amino acid sequence SGTLGHPGSLDETTYER (SEQ ID NO: 13) or composed thereof; and b) a peptide comprising the amino acid sequence LGGDLGTYVINK (SEQ ID NO: 14) or composed thereof; wherein the method comprises purifying the unprocessed TAT-GG-FXN from the sample by immunocapture prior to MS; wherein immunocapture is performed using a binding protein, such as an antibody, comprising an antigen-binding domain capable of binding a TAT protein transduction domain. In some embodiments, the binding protein, such as an antibody, comprises an antigen-binding domain that specifically binds to the TAT protein transduction domain present in TAT-GG-FXN.

[0153] In some aspects, this disclosure provides a method for determining the amount of FXN in a tissue sample, the method comprising determining the amount of at least one peptide derived from the FXN in the sample by means of MS, wherein the at least one peptide is selected from: a) a peptide comprising or consisting of the amino acid sequence SGTLGHPGSLDETTYER (SEQ ID NO: 13); and b) a peptide comprising or consisting of the amino acid sequence LGGDLGTYVINK (SEQ ID NO: 14); wherein the method is characterized by at least one of the following: the method has an accuracy of 20% or less as measured by % coefficient of variation (%CV); and the method has an accuracy of about 80% to about 120%.

[0154] Samples analyzed using the methods of this disclosure

[0155] Any sample can be used with the methods of this disclosure to determine the amount of FXN or FXN fusion protein, such as TAT-GG-FXN (SEQ ID NO: 11). In some embodiments, the methods of this disclosure can be used to analyze the amount of FXN or FXN fusion protein in solid samples, such as tissue samples, like oral swabs or skin biopsy samples. In some embodiments, the methods of this disclosure can be used to analyze the amount of FXN or FXN fusion protein in blood-derived samples, and may contain, for example, platelets. In some embodiments, the sample may be a plasma sample.

[0156] Oral swabs can be obtained from a subject, such as a mammal, like a monkey or human, by collecting oral cells using an isospiral swab. Oral swabs can be stored in cryovials until further use. Oral swabs can be further processed by adding a buffer such as RIPA buffer. In some embodiments, the RIPA buffer may also contain a protease inhibitor, such as EDTA-free Halt. TM A mixture of protease inhibitors. Oral swabs can be further vortexed and sonicated prior to immune capture.

[0157] Skin biopsies can be obtained from a subject, such as a mammal, like a monkey or a human, by collecting a biopsy puncture. The biopsy puncture can be rapidly frozen in liquid nitrogen until further use. Skin biopsies can be further processed by shredding the skin puncture, for example using a scalpel, and transferring the shredded tissue to a lysis matrix D-tube (MP Biomedicals). Skin biopsies can be further processed by adding a buffer, such as RIPA buffer. In some instances, RIPA buffer may also contain protease inhibitors, such as EDTA-free Halt. TM A mixture of protease inhibitors. Skin biopsies can be further subjected to ultrasound and homogenization, for example, using FastPrep-96. TM Grinding machine. The sonication and homogenization steps can be repeated as needed before immune capture.

[0158] In some embodiments, the methods of this disclosure are particularly suitable for analyzing solid samples, such as tissue samples, like oral swabs or skin biopsies, and are characterized by higher accuracy and precision compared to previously described methods for determining the amount of FXN. For example, previously described methods for determining the amount of FXN in samples such as solid tissues like oral swabs are characterized by high variability.

[0159] Furthermore, skin samples have not previously been used to measure FXN levels in subjects. The inventors unexpectedly and surprisingly discovered that FXN can be reliably measured in skin samples. The inventors also unexpectedly and surprisingly discovered that the level of FXN or FXN fusion proteins (such as TAT-GG-FXN (SEQ ID NO: 11)) in the skin is detectably increased due to the administration of FXN protein replacement therapy, such as a therapy including the administration of TAT-GG-FXN. Therefore, the inventors have discovered that skin samples, such as skin biopsies, can be used to monitor FXN levels in subjects, for example, after the administration of FXN or FXN fusion proteins such as TAT-GG-FXN.

[0160] Therefore, in some aspects, this disclosure provides a method for determining the amount of FXN in a skin sample, the method comprising determining the amount of at least one peptide derived from the FXN in the skin sample by using MS, wherein the at least one peptide is selected from: a) a peptide comprising the amino acid sequence GGMWTLGR (SEQ ID NO: 12); b) a peptide comprising the amino acid sequence SGTLGHPGSLDETTYER (SEQ ID NO: 13); and c) a peptide comprising the amino acid sequence LGGDLGTYVINK (SEQ ID NO: 14). In some embodiments, this disclosure provides a method for determining the amount of FXN in a skin sample, the method comprising determining the amount of at least one peptide derived from the FXN in the skin sample by using MS, wherein the at least one peptide is selected from: a) a peptide comprising the amino acid sequence GGMWTLGR (SEQ ID NO: 12); b) a peptide comprising the amino acid sequence SGTLGHPGSLDETTYER (SEQ ID NO: 13); and c) a peptide comprising the amino acid sequence LGGDLGTYVINK (SEQ ID NO: 14).

[0161] Example

[0162] Example 1

[0163] The purpose of this experiment was to develop an LC / MS / MS assay to determine the amount of TAT-GG-FXN (SEQ ID NO: 11) in oral cells, skin biopsies, and platelets from cynomolgus monkeys after repeated administration of TAT-GG-FXN for 14 days.

[0164] Materials and methods

[0165] biological samples

[0166] Preparation of biological samples used in the experiment, such as Figure 1 As shown.

[0167] Skin biopsy samples were prepared by shredding the skin through an 8mm skin perforation with a scalpel, homogenizing it in RIPA buffer using FastPrep-6, and then sonicating. Oral cell samples were prepared by extracting cells scraped from an oral swab (Isohelix SK-2S), lysing them by incubation in RIPA buffer, vortexing, and sonication. Platelet samples were prepared by collecting blood samples in 8.5mL ACD tubes, separating platelets by centrifugation, and lysing them in RIPA buffer by vortexing and sonication.

[0168] Calibration standards and QCs were prepared in TAT-GG-FXN-enhanced proxy matrices ranging from 0.250 to 25,000 ng / mL. Matrix QCs were prepared in pooled tissue homogenates at both low and high QC concentrations.

[0169] Total protein assay

[0170] Using Pierce TM The BCA Protein Assay Kit measures the total protein concentration of each sample and is used for data normalization.

[0171] Sample processing

[0172] Sample preparation for hybrid LC-MS / MS analysis, such as Figure 2 As shown, TAT-GG-FXN was purified by immunopurification using a biotinylated anti-FXN antibody. 15 N-labeled SILAC TAT-GG-FXN was used as an internal standard. Following trypsin digestion, the three peptides and their corresponding [specific parameters] shown in Table 4 were monitored by LC-MS / MS on a SCIEX 6500+. 15 N-labeled peptides.

[0173] Table 4. Monitored peptides used for TAT-GG-FXN quantification

[0174]

[0175] Result Processing

[0176] TAT-GG-FXN-derived trypsin peptides and their corresponding... 15 The peak area ratio of N-labeled peptides was used to construct the calibration curve (weighted 1 / x). 2 Linear regression).

[0177] The concentration of TAT-GG-FXN in the sample (in ng / mL) was determined using the calibration curve equation. After normalization using the total protein concentration (μg protein / mL homogenate), the TAT-GG-FXN concentration was reported as pg TAT-GG-FXN / μg total protein.

[0178] result

[0179] Table 5 below shows the results of TAT-GG-FXN measurements in oral cells, skin biopsies, and platelets. Table 5 also shows the precision and accuracy of the assays (peptides).

[0180] Table 5. Precision and accuracy of TAT-GG-FXN in oral cells, skin biopsies, and platelets (peptides)

[0181]

[0182]

[0183] ULOQ: Above the upper limit of quantification. Extrapolated values ​​are given.

[0184] *Intrinsic levels calculated in reverse. Average of 3 replicates.

[0185] **Endogenous levels at low and high QC levels + doped with TAT-GG-FXN.

[0186] Figure 3 This is a series of graphs showing the concentration of TAT-GG-FXN in monkey tissues (N=6) after repeated subcutaneous (SC) administration of 15 mg / kg BID for 14 days. Data are expressed as pg TAT-GG-FXN / μg total protein.

[0187] Figure 4 These are a series of representative chromatograms of TAT-GG-FXN trypsin peptide in monkey tissues before administration (day 1) and after repeated SC administration at 15 mg / kg BID for 14 days.

[0188] in conclusion

[0189] The results presented in this embodiment demonstrate that after repeated administration of TAT-GG-FXN (SEQ ID NO: 11) for 14 days, TAT-GG-FX not only accumulates in tissues outside the systemic circulation but also exists primarily as mature FXN protein. These results illustrate how mixed LC-MS / MS assays can be used to simultaneously understand the concentration, disposal, and processing of biotherapeutic agents, particularly TAT-GG-FXN.

Claims

1. A method for determining the amount of unprocessed fusion protein (FXN) in a sample, the method comprising determining the amount of peptide in the sample by mass spectrometry (MS). The unprocessed FXN fusion protein comprises a full-length FXN having the sequence SEQ ID NO: 1 and a cell-penetrating peptide (CPP) selected from HIV-TAT having the sequence SEQ ID NO: 4 and HIV-TAT+M having the sequence SEQ ID NO:

15. The peptide described herein consists of the amino acid sequence SGTLGHPGSLDETTYER (SEQ ID NO: 13); The method described therein includes purifying the unprocessed FXN fusion protein from the sample by immune capture prior to MS; The immune capture is performed using a binding protein containing an antigen-binding domain capable of binding HIV-TAT.

2. The method of claim 1, wherein the sample is obtained from a subject who has been administered the unprocessed FXN fusion protein.

3. The method of claim 1 or 2, wherein the method comprises the following steps: a) Purify the unprocessed FXN fusion protein from the sample by immunocapture to obtain an immunocapture complex containing the unprocessed FXN fusion protein; b) Digesting the immune-capture complex comprising the unprocessed FXN fusion protein with trypsin to produce a peptide consisting of SEQ ID NO: 13; and c) The amount of the peptide consisting of SEQ ID NO: 13 was determined by liquid chromatography and tandem mass spectrometry (LC / MS-MS).

4. The method of claim 3, wherein the liquid chromatography (LC) in step c) comprises reversed-phase chromatography.

5. The method of claim 3, wherein the MS-MS comprises monitoring the transition 607.3→669.3 of the peptide composed of SEQ ID NO:

13.

6. The method of claim 1 or 2, wherein the sample is a tissue sample.

7. The method of claim 6, wherein the tissue sample comprises an oral swab or a skin biopsy.

8. The method of claim 1 or 2, wherein the sample is derived from the blood of the subject.

9. The method of claim 8, wherein the sample comprises platelets or the sample is a plasma sample.

10. The method of claim 1 or 2, wherein the method has an accuracy of 20% or less, as measured by % coefficient of variation (%CV).

11. The method of claim 1 or 2, wherein the method has an accuracy of about 80% to about 120%.

12. The method of claim 1 or 2, wherein the method is linear for the concentration of unprocessed TAT-GG-FXN ranging from about 0.250 ng / mL to about 25.000 ng / mL.

13. A method for determining the amount of a common factor protein (FXN) in a tissue sample, the method comprising determining the amount of peptides derived from said FXN in said sample by using mass spectrometry (MS). The peptide described herein consists of the amino acid sequence SGTLGHPGSLDETTYER (SEQ ID NO: 13); Tissue samples include oral swabs or skin biopsies; The method is characterized by at least one of the following: The method has an accuracy of 20% or less, as measured by the % coefficient of variation (%CV); and The method has an accuracy of approximately 80% to approximately 120%. The method described therein includes purifying FXN from the sample by immune capture prior to MS.

14. The method of claim 13, wherein the sample is obtained from the subject.

15. The method of claim 14, wherein the subject is administered unprocessed FXN fusion protein.

16. The method of any one of claims 1, 2, and 15, wherein the unprocessed FXN fusion protein comprises, from the N-terminus to the C-terminus, a cell-penetrating peptide (CPP), a mitochondrial targeting sequence (MTS) of SEQ ID NO: 3, and FXN.

17. The method of claim 16, wherein the CPP comprises HIV-TAT (SEQ ID NO: 4) or HIV-TAT+M (SEQ ID NO: 15).

18. The method of claim 17, wherein the unprocessed FXN fusion protein is TAT-GG-FXN having the amino acid sequence of SEQ ID NO:

11.

19. The method of any one of claims 13-15, wherein the method comprises the following steps: a) Purify FXN from the sample by immunocapture to obtain an immunocapture complex containing FXN; b) Digesting the immune-capture complex containing FXN with trypsin to produce a peptide consisting of SEQ ID NO: 13; and c) The amount of peptides selected from the composition of SEQ ID NO: 13 was determined by liquid chromatography and tandem mass spectrometry (LC / MS-MS).

20. The method of claim 19, wherein in step c), the liquid chromatography (LC) comprises reversed-phase chromatography.

21. The method of claim 19, wherein the tandem mass spectrometry (MS-MS) includes monitoring the transition 607.3→669.3 of the peptide composed of SEQ ID NO:

13.

22. The method of any one of claims 13-15, wherein the immune capture is performed using a binding protein comprising an antigen-binding domain that specifically binds to FXN.

23. The method of claim 22, wherein the FXN is part of an FXN fusion protein further comprising HIV-TAT (SEQ ID NO: 4) or HIV-TAT+M (SEQ ID NO: 15), and wherein the binding protein specifically binds to the HIV-TAT.

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