IP Library › Granted Patent US 12,617,815
Granted Patent B2
US 12,617,815 · App. 17/776,478 · Granted May 5, 2026

Barcoded XTEN polypeptides and compositions thereof, and methods for making and using the same

Inventors: Volker Schellenberger (Palo Alto, CA); Eric Johansen (Oakland, CA); Angela Henkensiefken (San Jose, CA)
Assignee: Amunix Pharmaceuticals, Inc.
C07K1/13C07K14/001C12N15/70G01N33/6848C12N2800/101G01N2333/95
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Quick Facts
Patent No.
US 12,617,815
App. No.
17/776,478
Granted
May 5, 2026
Kind
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.

Claims (26)

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.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded May 10, 2024
From: SCHELLENBERGER, VOLKER; JOHANSEN, ERIC; HENKENSIEFKEN, ANGELA
To: AMUNIX PHARMACEUTICALS, INC.
Reel/Frame 067379/0339 →
Continuity (2)
Provisional Application 62934980 · Nov 13, 2019
Related Publication 20230287040A1 · Sep 14, 2023
References Cited (120)
US 4816567A · Schmuel et al. · 1989 [cited by applicant]
US 4826763A · Kjeld et al. · 1989 [cited by applicant]
US 5075222A · Hannum et al. · 1991 [cited by applicant]
US 5114923A · Seilhamer et al. · 1992 [cited by applicant]
US 5118666A · Habener · 1992 [cited by applicant]
US 5175146A · Basava et al. · 1992 [cited by applicant]
US 5234906A · Young et al. · 1993 [cited by applicant]
US 5324639A · Brierley et al. · 1994 [cited by applicant]
US 5364840A · Basava et al. · 1994 [cited by applicant]
US 5364934A · Drayna et al. · 1994 [cited by applicant]
US 5374618A · Craig et al. · 1994 [cited by applicant]
US 5424286A · Eng · 1995 [cited by applicant]
US 5521283A · Dimarchi et al. · 1996 [cited by applicant]
US 5532336A · Dimarchi et al. · 1996 [cited by applicant]
US 5534617A · Cunningham et al. · 1996 [cited by applicant]
US 5574010A · McFadden · 1996 [cited by applicant]
US 5604203A · Balasubramaniam et al. · 1997 [cited by applicant]
US 5631230A · Fang et al. · 1997 [cited by applicant]
US 5674710A · Seilhamer et al. · 1997 [cited by applicant]
US 5686411A · Gaeta et al. · 1997 [cited by applicant]
US 5821337A · Carter et al. · 1998 [cited by applicant]
US 5843446A · Ladd et al. · 1998 [cited by applicant]
US 5885573A · Bluestone et al. · 1999 [cited by applicant]
US 5948761A · Seilhamer et al. · 1999 [cited by applicant]
US 6214797B1 · Vale et al. · 2001 [cited by applicant]
US 6320022B1 · Cuttitta et al. · 2001 [cited by applicant]
US 6406632B1 · Safir et al. · 2002 [cited by applicant]
US 6491916B1 · Bluestone et al. · 2002 [cited by applicant]
US 6716626B1 · Itoh et al. · 2004 [cited by applicant]
US 6806063B2 · Pedersen et al. · 2004 [cited by applicant]
US 6858409B1 · Thompson et al. · 2005 [cited by applicant]
US 6965013B2 · Hsu · 2005 [cited by applicant]
US 6967237B2 · Bednarek · 2005 [cited by applicant]
US 7112659B2 · Mann et al. · 2006 [cited by applicant]
US 7166575B2 · Quay · 2007 [cited by applicant]
US 7271238B2 · Gaeta et al. · 2007 [cited by applicant]
US 7294513B2 · Wyatt · 2007 [cited by applicant]
US 7385026B1 · Kangawa et al. · 2008 [cited by applicant]
US 8044175B2 · Dransfield et al. · 2011 [cited by applicant]
US 8067532B2 · Maclean · 2011 [cited by applicant]
US 20010027181A1 · Kitakaze et al. · 2001 [cited by applicant]
US 20020042367A1 · Stewart et al. · 2002 [cited by applicant]
US 20020119490A1 · Martinez-Martin et al. · 2002 [cited by applicant]
US 20030228309A1 · Salcedo et al. · 2003 [cited by applicant]
US 20080261886A1 · Hedner · 2008 [cited by applicant]
US 20100239554A1 · Schellenberger et al. · 2010 [cited by applicant]
US 20100323956A1 · Schellenberger et al. · 2010 [cited by applicant]
US 20110046060A1 · Schellenberger et al. · 2011 [cited by applicant]
US 20110046061A1 · Schellenberger et al. · 2011 [cited by applicant]
US 20110077199A1 · Schellenberger et al. · 2011 [cited by applicant]
US 20110172146A1 · Schellenberger et al. · 2011 [cited by applicant]
US 20120034228A1 · Kufer et al. · 2012 [cited by applicant]
US 20180030531A1 · de Raad et al. · 2018 [cited by applicant]
US 20190024145A1 · Vigneault et al. · 2019 [cited by applicant]
EP 4058466A1 · 2022 [cited by applicant]
WO WO1987001728A1 · 1987 [cited by applicant]
WO WO1996022308A2 · 1996 [cited by applicant]
WO WO1998052976A1 · 1998 [cited by applicant]
WO WO2000003317A1 · 2000 [cited by applicant]
WO WO2000057183A1 · 2000 [cited by applicant]
WO WO2002079232A2 · 2002 [cited by applicant]
WO WO2005118635A2 · 2005 [cited by applicant]
WO WO2007033230A2 · 2007 [cited by applicant]
WO WO2010091122A1 · 2010 [cited by applicant]
WO WO2010144502A2 · 2010 [cited by applicant]
WO WO2010144508A1 · 2010 [cited by applicant]
WO WO2011028228A1 · 2011 [cited by applicant]
WO WO2011028229A1 · 2011 [cited by applicant]
WO WO2011028344A2 · 2011 [cited by applicant]
WO WO2014011819A2 · 2014 [cited by applicant]
WO WO2015023891A2 · 2015 [cited by applicant]
WO WO2016077505A2 · 2016 [cited by applicant]
WO WO2016145416A2 · 2016 [cited by examiner]
WO 2019074841A1 · 2019 [cited by applicant]
WO WO2019126576A1 · 2019 [cited by applicant]
WO WO2021097186A1 · 2021 [cited by applicant]
Balzar et al, “The biology of the 17-1A antigen (Ep-CAM)”, Journal of Molecular Medicine, Oct. 1999, vol. 77, No. 10, pp. 699-712. [cited by applicant]
Banner et al., “Crystal structure of the soluble human 55 kd TNF receptor-human TNFβ complex: Implications for TNF receptor activation”, Cell, May 7, 1993, 73(3): 431. [cited by applicant]
Becker et al. “Clinical Review 167: Procalcitonin and the Calcitonin Gene Family of Peptides in Inflammation, Infection, and Sepsis: A Journey from Calcitonin Back to Its Precursors”, JCEM, Apr. 2004, vol. 89, No. 4, pp… [cited by applicant]
Bell, “Isolation of the human insulin-like growth factor genes: Insulin-like growth factor II and insulin genes are contiguous”, Proc Natl Acad Sci USA., Oct. 1985, vol. 82, pp. 6450-6454. [cited by applicant]
Buchanan et al., “Engineering a therapeutic IgG molecule to address cysteinylation, aggregation and enhance thermal stability and expression”, mAbs, Mar. 1, 2013, Landes Bioscience, vol. 5, Nr: 2, pp. 255-262. [cited by applicant]
Burgess et al., “The heparin-binding (fibroblast) growth factor family of proteins”, Ann. Rev. Biochem., 1989, vol. 58, pp. 575-606. [cited by applicant]
Caliceti et al., “Pharmacokinetic and Biodistribution Properties of Poly(ethylene glycol)-Protein Conjugates”, Advanced Drug Delivery Reviews, Sep. 26, 2003, vol. 55, pp. 1261-1277. [cited by applicant]
Chatenoud, “CD3-specific antibody-induced active tolerance: from bench to bedside”, Nat. Rev. Immunol., Feb. 1, 2003, vol. 3, pp. 123-132. [cited by applicant]
Chen et al., Influence of Histidine on the Stability and Physical Properties of a Fully Human Antibody in Aqueous and Solid Forms, Pharmaceutical Research, 2003, vol. 20, pp. 1952-1960. [cited by applicant]
Chou et al., “Prediction of protein conformation”, Jan. 15, 1974, Biochemistry, vol. 13, Iss. 2, pp. 222-245. [cited by applicant]
Couder et al., Int. J. Peptide Protein Res., (19930000), vol. 41, pp. 181-184. [cited by applicant]
Coulier et al., “The human and mouse fibroblast growth factor 6 (FGF6) genes and their products: possible implication in muscle development”. Prog. Growth Factor Res., 1994, vol. 5, p. 1. [cited by applicant]
Fu et al., Endocrinology, 2004, vol. 145, pp. 2504-2603. [cited by applicant]
Garnier et al., “GOR method for predicting protein secondary structure from amino acid sequence”, Methods Enzymol, 1996, vol. 266, pp. 540-553. [cited by applicant]
Gastl, “Ep-CAM overexperssion in breast cancer as a predictor of survival”, Lancet, Dec. 9, 2000, vol. 356, Issue 9246, pp. 1981-1982. [cited by applicant]
George et al., “An analysis of protein domain linkers: their classification and role in protein folding”, Protein Engineering, 2003, vol. 15, No. 11, pp. 871-879. [cited by applicant]
Ghirlando et al., “Glycosylation of human IgG-Fc: influences on structure revealed by differential scanning micro-calorimetry”, Immunology Letters, May 3, 1999, vol. 68, issue 1, pp. 47-52. [cited by applicant]
Herold et al., “Anti-CD3 monoclonal antibody in new-onset type1 diabetes mellitus”, New England Journal of Medicine, May 1, 2002, vol. 346, pp. 1692-1698. [cited by applicant]
International Search Report and Written Opinion for PCT International Patent Application No. PCT/2020/060378, mailed Mar. 1, 2021. [cited by applicant]
Ishimitsu et al., “Genomic Structure of Human Adrenomedullin Gene”, Biochem. Biophys. Res. Commun, Aug. 30, 1994, vol. 203, Issue1, pp. 631-639. [cited by applicant]
Kangawa et al., “Identification in Rat Atrial Tissue of Multiple Forms of Natriuretic Polypeptides of About 3,000 Daltons”, BBRC, Jun. 15, 1984, vol. 121, issue 2, p. 585. [cited by applicant]
Kangawa et al., “Purification and Complete Amino Acid Sequence of α-human Atrial Natriuretic Polypeptide (α-hANP)”, Biochemical and Biophysical Research Communications, Jan. 13, 1984, vol. 118, No. 1, pp. 131-139. [cited by applicant]
Koda, “Amylin concentrations and glucose control”, Lancet, May 9, 1992, vol. 339, issue 8802, pp. 1179-1180. [cited by applicant]
Köhler et al., “Continuous cultures of fused cells secreting antibody of predefined specificity”, Nature, Aug. 1, 1975, vol. 256, p. 495. [cited by applicant]
Kojima et al., “Ghrelin is a growth-hormone-releasing acylated peptide from stomach”, Nature, Dec. 9, 1999, vol. 402, pp. 656-660. [cited by applicant]
Kumar et al., ATGAM versus OKT3 induction therapy in cadaveric kidney transplantation: patient and graft survival, CD3 subset, infection, and cost analysis, Transplant. Proc., 1998, vol. 30, issue 4, pp. 1351-1352. [cited by applicant]
Marins et al., “A growth hormone-based phylogenetic analysis of euteleostean fishes including a representative species of the Atheriniformes Order, Odontesthes argentinensis”, Genetics and Molecular Biology, 2003, vol. … [cited by applicant]
Martin et al., “Expression of the 17-1A antigen in gastric and gastro-esophageal junction adenocarcinomas: a potential immunotherapeutic target?”, J Clin Pathol, 1999, vol. 52, pp. 701-704. [cited by applicant]
Miyamoto et al., “Peptide barcoding for establishment of new types of genotype-phenotype linkages”, Publication data: PLOS One, Apr. 23, 2019, Public Library of Science, vol. 14, No. 4, pp. e0215993. [cited by applicant]
Murray et al., “Epitope Affinity Chromatography and Biophysical Studies of Monoclonal Antibodies and Recombinant Antibody Fragments”, Journal of Chromatography Science, 2002, vol. 40, pp. 343-349. [cited by applicant]
Packeisen et al., “Detection of Surface Antigen 17-1A in Breast and Colorectal Cancer”, Apr. 25, 1999, vol. 18, Nr: 1, pp. 37-40. [cited by applicant]
Passlick et al., “The 17-1A antigen is expressed on primary, metastatic and disseminated non-small cell lung carcinoma cells”, International Journal of Cancer, Aug. 15, 2000, vol. 87, No. 4, pp. 548-552. [cited by applicant]
Rafaeloff et al., “Cloning and sequencing of the pancreatic islet neogenesis associated protein (INGAP) gene and its expression in islet neogenesis in hamsters”, J Clin Invest, May 1, 1997, vol. 99, No. 9, pp. 2100-2109. [cited by applicant]
Rawlings et al., “MEROPS: the peptidase database”, Nucleic Acids Res., 2008, vol. 36, Database issue, p. D320. [cited by applicant]
Sgro, “Side-effects of a monoclonal antibody, muromonab CD3/orthoclone OKT3: bibliographic review”, Toxicology, Dec. 20, 1995, vol. 105, Issue 1, pp. 23-29. [cited by applicant]
Shalaby et al., “Development of humanized bispecific antibodies reactive with cytotoxic lymphocytes and tumor cells overexpressing the HER2 protooncogene”, J. Exp. Med., Jan. 1, 1992, vol. 175, Issue 1, pp. 217-225. [cited by applicant]
Smith et al., “Nonmitogenic anti-CD3 monoclonal antibodies deliver a partial T cell receptor signal and induce clonal anergy”, Publication data: Journal of Experimental Medicine,,Apr. 21, 1997, vol. 185, No. 8, pp. 1413… [cited by applicant]
Stickler et al., “Human population-based identification of CD4(+) T-cell peptide epitope determinants”, J Immunol Methods, 2003, vol. 281, pp. 95-108. [cited by applicant]
Sturniolo, T. et al., “Generation of tissue-specific and promiscuous HLA ligand databases using DNA microarrays and virtual HLA class II matrices”, Nat Biotechnol, Jun. 1999, vol. 17, pp. 555-561. [cited by applicant]
Sudoh et al., “Cloning and sequence analysis of cDNA encoding a precursor for human brain natriuretic peptide”, BBRC,, Mar. 31, 1989, vol. 159, issue 3, p. 14207-1434. [cited by applicant]
Tunnacliffe, “The majority of human CD3 epitopes are conferred by the epsilon chain”, International Immunology, Nov. 1989, vol. 1, issue 5, pp. 546-550. [cited by applicant]
Van Zoelen et al., “The use of nonhomologous Scratchard analysis in the evaluation of ligand-protein interactions”, Trends Pharmacol Sciences, Dec. 1, 1998, vol. 19, No. 12, p. 487-490. [cited by applicant]
Wong, “The mechanism of anti-CD3 monoclonal antibodies. Mediation of cytolysis by inter-T cell bridging”, Transplantation, Oct. 1990, vol. 50, pp. 683-689. [cited by applicant]
Egloff, et al. Engineered peptide barcodes for in-depth analyses of binding protein libraries. Nature Methods. Apr. 22, 2019; 16(5):421-428. with Supplementary information. [cited by applicant]