US-12617815-B2 - Barcoded XTEN polypeptides and compositions thereof, and methods for making and using the same

US12617815B2US 12617815 B2US12617815 B2US 12617815B2US-12617815-B2

Abstract

Disclosed herein are polypeptides comprising an extended recombinant polypeptide (XTEN) comprised of a plurality of overlapping sequence motifs and one or more barcode fragments releasable upon protease digestion and detectable from ail other proteolytic-ally releasable fragments. Certain embodiments of these polypeptides further comprise a biologically active polypeptide, wherein advantageous embodiments thereof comprise a releasable segment capable of proteolytic cleavage that cleaves the linkage between the XTEN polypeptide and the biologically active polypeptide. Methods of making and methods of using said polypeptides are also disclosed.

Inventors

Volker Schellenberger
Eric Johansen
Angela HENKENSIEFKEN

Assignees

AMUNIX PHARMACEUTICALS, INC.

Dates

Publication Date: 20260505
Application Date: 20201113

Claims (12)

1 . A method of detecting truncation of a fusion polypeptide in a sample, wherein the fusion polypeptide comprises at least one extended recombinant (XTEN) polypeptide fused to the N-terminus or the C-terminus of a biologically active polypeptide comprising a reference fragment, wherein the XTEN polypeptide is from 150 to 1,000 amino acids in length, comprises a plurality of non-overlapping sequence motifs each between 9 and 14 amino acids in length, and at least 90% of the amino acid residues of the XTEN polypeptide are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) or proline (P), wherein the XTEN comprises a barcode fragment, which is from 4 to 20 amino acids in length, occurs only once in the fusion polypeptide, and is located within 100 amino acids from the N-terminus of the XTEN polypeptide if the XTEN polypeptide is N-terminal to the biologically active polypeptide or from the C-terminus of the XTEN polypeptide if the XTEN polypeptide is C-terminal to the biologically active polypeptide, wherein the sample comprises a first set of polypeptides and a second set of polypeptides, wherein each polypeptide of the first set of polypeptides (a) has the same sequence as, or is truncated from, the fusion polypeptide and (b) retains the barcode fragment, and each polypeptide of the second set of polypeptides (a) is truncated from the fusion polypeptide and (b) lacks the barcode fragment, wherein each polypeptide of both the first set of polypeptides and the second set of polypeptides retains the reference fragment, the method comprising: contacting the sample with a protease to produce a plurality of proteolytic fragments that result from cleavage of the first set of polypeptides and the second set of polypeptides, wherein the plurality of proteolytic fragments comprise: a plurality of reference fragments; and a plurality of barcode fragments, and determining a ratio of the amount of barcode fragments to the amount of reference fragments, thereby assessing the relative amounts of the first set of polypeptides to the second set of polypeptides representing the truncation of the fusion polypeptide.
2 . The method of claim 1 , wherein the XTEN polypeptide has at least 90% sequence identity to a sequence selected from SEQ ID NOs: 8001-8019.
3 . The method of claim 1 , wherein the barcode fragment comprises a sequence having at least 90% sequence identity to one selected from SEQ ID NO: 8020-8030.
4 . The method of claim 1 , wherein the fusion polypeptide further comprises a second XTEN polypeptide on the other side of the biologically active polypeptide, wherein the second XTEN polypeptide is from 150 to 1,000 amino acids in length, comprises a plurality of non-overlapping sequence motifs each between 9 and 14 amino acids in length, and at least 90% of the amino acid residues of the second XTEN polypeptide are glycine (G), alanine (A), serine(S), threonine (T), glutamate (E) or proline (P), and wherein the second XTEN polypeptide comprises a second barcode fragment which is from 4 to 20 amino acids in length, occurs only once in the fusion polypeptide, and is located within 100 amino acids from the C-terminus of the second XTEN polypeptide if the second XTEN polypeptide is C-terminal to the biologically active polypeptide or from the N-terminus of the second XTEN polypeptide if the second XTEN polypeptide is N-terminal to the biologically active polypeptide.
5 . The method of claim 4 , wherein the second XTEN polypeptide has at least 90% sequence identity to a sequence selected from SEQ ID NOs: 8001-8019.
6 . The method of claim 4 , wherein the second barcode fragment comprises a sequence having at least 90% sequence identity to one selected from SEQ ID NO: 8020-8030.
7 . The method of claim 1 , wherein the protease is a Glu-C protease.
8 . The method of claim 1 , wherein the protease is not trypsin.
9 . The method of claim 1 , wherein determining a ratio of the amount of barcode fragments to the amount of reference fragments comprises quantifying barcode fragments and reference fragments from the sample after it has been contacted with the protease.
10 . The method of claim 9 , wherein the barcode fragments and the reference fragments are identified based on their respective masses.
11 . The method of claim 1 , wherein the barcode fragments and the reference fragments are identified via: (i) mass spectrometry, and/or (ii) liquid chromatography-mass spectrometry (LC-MS).
12 . The method of claim 1 , wherein determining a ratio of the barcode fragments to the reference fragments comprises: (i) isobaric labeling, and/or (ii) spiking the sample with one or both of an isotope labeled reference fragment and an isotope labeled barcode fragment.

Description

RELATED APPLICATIONS This application is a 35 U.S.C. § 371 filing of International Patent Application No. PCT/US2020/060378, filed Nov. 13, 2020, which claims priority to U.S. Provisional Patent Application Ser. No. 62/934,980, the entire disclosures of which are hereby incorporated herein by reference. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Nov. 6, 2020, is named 20-1761-WO_Sequence_Listing_ST25.txt and is 1494 bytes in size. BACKGROUND A polypeptide can be produced in a manner that results in a mixture of polypeptides. The mixture of polypeptides can often include the full-length polypeptide, along with size variants (e.g., truncations) thereof. The presence of variants that differ in size from the desired full-length product can affect the biological behavior of a polypeptide drug substance, potentially affecting the safety and/or efficacy of the polypeptide drug substance. For example, protein-based prodrugs for cancer therapy can be engineered with a tumor-targeted activation mechanism. More specifically, the full-length therapeutic protein can be produced and administered in an inactive (non-cytotoxic) prodrug form, that is converted to the active drug by preferential removal of a portion of the prodrug polypeptide at the intended biological side (e.g., the tumor). Truncation variants of the full-length construct can lose protective sequences and become cytotoxic (active), thus “contaminating” the prodrug composition and producing a mixture having components that are unintentionally active outside the intended biological site. In some instances, such shorter length variants can pose a greater risk of immunogenicity, have less selective toxicity for tumor cells, or show a less desired pharmacokinetic profile (e.g., resulting in a narrowed therapeutic window) compared with the full-length protein, or deleteriously have unintended effects in a recipient outside the intended site (e.g. in healthy tissue). As a result, detection and quantification of protein structural variations can be important for assessing biological properties (e.g., clinical safety and pharmacologic efficacy) of biotherapeutics and in developing new biotherapeutics (e.g., with increased efficacy and reduced side effects). Existing techniques and methods for identifying and quantifying the amount of “contaminating” truncation products can include one or more drawbacks, such as being of limited sensitivity, ease, efficiency, or effectiveness. SUMMARY Disclosed herein are polypeptides comprising an extended recombinant polypeptide (XTEN) that is comprised of a plurality of non-overlapping sequence motifs. In XTEN polypeptides of this invention, the plurality of non-overlapping sequence motifs comprise: a set of non-overlapping sequence motifs, wherein each of said sequence motifs is repeated at least twice in the XTEN polypeptide; and also a unique non-overlapping sequence motif that occurs only once within the XTEN polypeptide; wherein the polypeptide further comprises a first barcode fragment releasable from the polypeptide upon digestion by a protease. In said embodiments, the first barcode fragment is a portion of the XTEN that includes at least part of the sequence motif that occurs only once within the XTEN and differs in sequence and molecular weight from all other peptides fragments that are releasable from the polypeptide upon complete digestion of the polypeptide by the protease. Further, in XTEN embodiments of the invention provided herein, the barcode fragment does not include the N-terminal amino acid or the C-terminal amino acid of the polypeptide. As further disclosed herein, XTEN polypeptides of this invention are characterized as comprising at least 150 amino acids, more specifically 150-3000 amino acids in length. The amino acids comprising XTEN polypeptides of the invention are characterized wherein at least 90% of these residues are glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), or proline (P), and the XTEN polypeptide comprises at least four of these amino acids (G, A, S, T, E, or P). In addition, XTEN polypeptides as provided herein comprise nonoverlapping sequence motifs that are 9 to 14 amino acid sequences in length and within each of said nonoverlapping motifs the sequence of G, A, S, T, E, or P amino acids is substantially randomized with respect to any other nonoverlapping sequence motif comprising the XTEN polypeptide. In some embodiments, the barcode fragment does not include a glutamic acid that is immediately adjacent to another glutamic acid in the XTEN. In some embodiments, the barcode fragment has a glutamic acid at its C-terminus. In some embodiments, the barcode fragment has an N-terminal amino acid that is immediately preceded by a glutamic acid residue. In some embodiments, the glutamic acid residue that prece