XTEN conjugate compositions and methods of making same

• US 10,172,953 B2
• Filed: 02/27/2013
• Issued: 01/08/2019
• Est. Priority Date: 02/27/2012
• Status: Active Grant

• Current Assignee: Amunix Pharmaceuticals, Inc. (Sanofi )
• Original Assignee: Amunix Operating Inc.
• Inventors: Schellenberger, Volker, Podust, Vladimir, Wang, Chia-Wei, McLaughlin, Bryant, Sim, Bee-Cheng, Ding, Sheng, Gu, Chen
• Primary Examiner(s): Beane, Randall L
• Application Number:
  US14/381,199
• Publication Number:
  US 20150037359A1
• Time in Patent Office: 2,141 Days
• CPC Class Codes: A61K 38/24 Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
• CPC Class Codes: A61K 47/64 Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent peptidic linkers A61K47/65
• CPC Class Codes: A61K 49/0032 Methine dyes, e.g. cyanine dyes
• CPC Class Codes: A61K 49/0052 Small organic molecules oligomers, polymers, dendrimers A61K49/0054
• CPC Class Codes: A61K 49/0056 Peptides, proteins, polyamino acids
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/04 Centrally acting analgesics, e.g. opioids
• CPC Class Codes: A61P 27/00 Drugs for disorders of the senses
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 5/00 Drugs for disorders of the endocrine system
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C07K 5/0205 : containing the structure -NH-(X)3-C(=0)-, e.g. statine or derivatives thereof

Abstract

The present invention relates to extended recombinant polypeptide (XTEN) compositions, conjugate compositions comprising XTEN and XTEN linked to cross-linkers useful for conjugation to pharmacologically active payloads, methods of making highly purified XTEN, methods of making XTEN-linker and XTEN-payload conjugates, and methods of using the XTEN-cross-linker and XTEN-payload compositions.

Claims

1. A composition comprising a homogeneous population of polypeptides comprising an extended recombinant polypeptide (XTEN), wherein at least 90% of individual polypeptide molecules in said population have an identical sequence length, wherein the XTEN is characterized in that:

(a) the XTEN is 36 to 3000 L-amino acid residues in length;

(b) the sum of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) residues constitutes more than 90% of the total amino acid residues of the XTEN; and

(c) the XTEN sequence comprises one or more sequence motifs selected from SEQ ID NOs;

26-55 and one or more sequence motifs selected from SEQ ID NOs;

399-407;

wherein the polypeptides of the population have a configuration of formula II;

(HS)-(CS1)-(XTEN)-(CS2)-(AT1) 

 

II

wherein

(i) HS is a sequence having at least 90% sequence identity to a sequence selected from SEQ ID NOs;

517-526;

(ii) AT1 is a first affinity tag having an amino acid sequence exhibiting binding affinity for a chromatography substrate selected from the group consisting of hydrophobic interaction chromatography (HIC) substrate, cation exchange substrate, anion exchange substrate, immobilized metal ion affinity chromatography (IMAC) substrate, and immobilized antibody substrate;

(iii) CS1 is a first amino acid sequence capable of being cleaved by trypsin;

(iv) CS2 is a second amino acid sequence capable of being cleaved by trypsin; and

(v) XTEN is the extended recombinant polypeptide.

2. The composition of claim 1, wherein the XTEN sequence has at least 90% sequence identity to a sequence selected from SEQ ID NOs:

193-205, 219-258, 267-279, 293-332, 341-350, 361, 362, 364-369, 373, 374, 376, 377, 379-385, and 387-391.

3. The composition of claim 1, wherein the XTEN sequence comprises at least 1 to 10 cysteine amino acids.

4. The composition of claim 1, wherein the AT1 is selected from SEQ ID NO:

18, SEQ ID NO;

20, and SEQ ID NO;

433-447.

5. The composition of claim 4, wherein the first chromatography substrate is IMAC or cation exchange substrate, and the AT1 comprises the sequence HHHHHH (SEQ ID NO:

18) or HHHHHHHH (SEQ ID NO;

     20).

6. The composition of claim 5, wherein the CS1 and the CS2 amino acid sequences independently comprise the sequence RX or KX, wherein X is any L-amino acid other than proline.

7. The composition of claim 5, wherein the CS1 and the CS2 amino acid sequences independently are selected from SEQ ID NO:

467, SEQ ID NO;

468, SEQ ID NO;

469, SEQ ID NO;

470, SEQ ID NO;

471, and SEQ ID NO;

472.

8. The composition of claim 7, wherein the CS1 and the CS2 amino acid sequences independently are SASRSA (SEQ ID NO:

467) or SASKSA (SEQ ID NO;

     468).

9. The composition of claim 6, wherein the polypeptides of the population further comprise a second affinity tag selected from SEQ ID NO:

18, SEQ ID NO;

20, and SEQ ID NOs;

443-447, and wherein the second affinity tag exhibits binding affinity for a chromatography substrate selected from the group consisting of hydrophobic interaction chromatography (HIC) substrate, cation exchange substrate, anion exchange substrate, immobilized metal ion affinity chromatography (IMAC) substrate, and immobilized antibody substrate.

10. A polynucleotide encoding the composition of claim 6.

11. A host cell comprising a vector comprising the polynucleotide of claim 10.

12. A method of producing a composition comprising a homogeneous population of polypeptides, the method comprising:

(a) providing the host cell of claim 11;

(b) culturing the host cell under conditions causing or permitting the population of polypeptides to be expressed in the host cell, thereby producing the population of polypeptides;

(c) adsorbing the expressed population of polypeptides onto a chromatography substrate under conditions effective to capture the AT1;

(d) treating said composition with trypsin under conditions effective to cleave the CS1 and CS2 amino acid sequences; and

(e) recovering the XTEN.

13. The method of claim 12, wherein at least 90% of the recovered XTEN have an identical sequence length.

14. The method of claim 12, wherein the chromatography substrate is selected from the group consisting of hydrophobic interaction chromatography (HIC) substrate, cation exchange substrate, anion exchange substrate, immobilized metal ion affinity chromatography (IMAC) substrate, and immobilized antibody substrate.

15. The method of claim 14, wherein the chromatography substrate is immobilized metal ion affinity chromatography (IMAC) substrate.

16. The method of claim 12 wherein the host cell is a prokaryotic cell.

17. The method of claim 12, further comprising attaching a first cross-linker to the recovered XTEN, wherein the first cross-linker is selected from an N-maleimide, an iodoacetyl reagent, a pyridyl disulfide reagent, a vinyl sulfone reagent, 3-propargyloxypropanoic acid, (oxyethyl)_n-acetylene where n is 1-10, dibenzylcyclooctyne (DBCO), cyclooctyne (COT), 3-azide-propionic acid, 6-azide-hexanoic acid, and (oxyethyl)n-azide, where n is 1-10.

18. The method of claim 17, wherein the first cross-linker is conjugated to the XTEN at a location selected from:

(a) an alpha-amino group of an N-terminal amino acid residue of the XTEN; and

(b) a thiol group of a cysteine residue of the XTEN.

19. The method of claim 17, wherein the first cross-linker is N-maleimide.

20. The method of claim 17, further comprising conjugating a first payload to the first cross-linker through a single atom residue of the first payload, wherein the residue is selected from the group consisting of carbon, nitrogen, oxygen and sulfur.

21. The method of claim 20, wherein the first payload is selected from doxorubicin, paclitaxel, monomethyl auristatin E, monomethyl auristatin F, maytansine, maytansinoid DM1, maytansinoid DM4, calicheamicin, desacetylvinblastine monohydrazide, camptothecin, mitomycin C, epothilone, hTNF, IL-12, bortezomib, ranpirnase, Pseudomonas exotoxin, SN-38, rachelmycin, m-TOR inhibitor, rapamycin, tubulysin B, tubulysin M, and duocarmycin.

22. The method of claim 17, further comprising conjugating a payload to the first cross-linker, wherein the payload is selected from doxorubicin, paclitaxel, monomethyl auristatin E, monomethyl auristatin F, maytansine, maytansinoid DM1, maytansinoid DM4, calicheamicin, desacetylvinblastine monohydrazide, camptothecin, mitomycin C, epothilone, hTNF, IL-12, bortezomib, ranpirnase, Pseudomonas exotoxin, SN-38, rachelmycin, m-TOR inhibitor, rapamycin, tubulysin B, tubulysin M, and duocarmycin.

23. The method of claim 22, further comprising conjugating a targeting moiety to the N-terminus of the XTEN, wherein the targeting moiety is a peptide, protein, antibody-like scaffold, antibody, or antibody fragment, and wherein the first cross-linker is conjugated to a thiol group of a cysteine residue of the XTEN.

24. The method of claim 23, wherein the targeting moiety is an antibody or antibody fragment.