IP Library › Granted Patent US 12,391,956
Granted Patent B2
US 12,391,956 · App. 16/931,266 · Granted Aug 19, 2025

Methods, compositions and cells for preparing surfactant protein D (SP-D)

Inventors: Jan Susan Rosenbaum (Cincinnati, OH); Frederick Gyapon Quast (Berlin, DE); Matthias Kaup (Berlin, DE); Lars Stöckl (Berlin, DE)
Assignees: Airway Therapeutics, Inc.; Glycotope GmbH
C12N15/85C07K14/7051C07K14/785C12N5/0694C12N9/003C12Y105/01003C07K2319/00C12N2015/8518
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Quick Facts
Patent No.
US 12,391,956
App. No.
16/931,266
Granted
Aug 19, 2025
Kind
B2
Abstract

Some embodiments of the methods and compositions provided herein relate to the preparation surfactant protein-D (SP-D). Some embodiments include the expression of human SP-D in certain cell lines, and the purification of human SP-D from such cell lines. Some embodiments include the preparation of certain oligomeric forms of human SP-D.

Claims (33)

1. A method for producing a human surfactant protein D (SP-D) polypeptide composition comprising:

(a) introducing a polynucleotide encoding the SP-D polypeptide into a human cell selected from the group consisting of NM-H9D8, NM-H9D8-E6Q12, NM-H9D8(8B11), and NM-F9;

(b) culturing the cell under conditions in which the SP-D polypeptide is expressed; and

(c) isolating the expressed SP-D polypeptide from the cell,

wherein

the expressed SP-D polypeptide has an antennarity number within a range from 190 to 215, or

an antennarity number for the expressed SP-D polypeptide and an antennarity number for a naturally occurring human SP-D are within 10% from each other.

2. The method of claim 1 , wherein an antennarity number for the expressed SP-D polypeptide and an antennarity number for a naturally occurring human SP-D are within 4% from each other.

3. The method of claim 1 , wherein a magnitude of a peak at 50 minutes in an N-glycosylation profile for the expressed SP-D polypeptide and a magnitude of a peak at 50 minutes in an N-glycosylation profile for a naturally occurring human SP-D are within 10% from each other.

4. The method of claim 1 , wherein the expressed SP-D polypeptide has an N-glycosylation profile comprising a peak at 50 minutes having a greater magnitude than a peak at 50 minutes in an N-glycosylation profile for a human SP-D polypeptide expressed from a non-human cell.

5. The method of claim 1 , wherein the expressed SP-D polypeptide has an N-glycosylation profile comprising a peak at 43 minutes having a greater magnitude than a peak at 43 minutes in an N-glycosylation profile for a human SP-D polypeptide expressed from a non-human cell.

6. The method of claim 1 , wherein the expressed SP-D polypeptide has an O-glycosylation profile comprising a peak at between 5 and 6 minutes having a greater magnitude than a peak at between 5 and 6 minutes in an O-glycosylation profile for a human SP-D polypeptide expressed from a non-human cell.

7. The method of claim 1 , wherein the cell is a NM-H9D8 cell.

8. The method of claim 1 , wherein the cell is a NM-H9D8(8B11) cell.

9. The method of claim 1 , wherein the polynucleotide encodes a leader polypeptide selected from a wild type SP-D polypeptide leader sequence, and a wild type T-cell receptor (TCR) polypeptide leader sequence.

10. The method of claim 1 , wherein the polynucleotide encodes a leader polypeptide comprising the amino acid sequence of SEQ ID NO:05 or SEQ ID NO: 10.

11. The method of claim 1 , wherein the polynucleotide encodes a pre-polypeptide comprising a leader polypeptide and the SP-D polypeptide, the pre-polypeptide comprising the amino acid sequence of SEQ ID NO:04 or SEQ ID NO:09.

12. The method of claim 1 , further comprising isolating a population of the expressed SP-D polypeptides, each expressed SP-D polypeptide comprising a complex-type carbohydrate attached at an N-glycosylation site, wherein the population has a glycosylation pattern comprising the following characteristics:

(i) at least 70% of the complex-type carbohydrates include a core fucose;

(ii) at least 10% of the complex-type carbohydrates include at least one sialic acid residue;

(iii) at least 50% of the complex-type carbohydrates include at least a biantennary carbohydrate structure;

(iv) at least 10% of the complex-type carbohydrates include a bisecting N-acetylglucosamine;

(v) less than 10% of the carbohydrates are high-mannose type structures; and

(vi) a detectable amount of α2,6-coupled sialic acid residues.

13. The method of claim 12 , wherein the population has a glycosylation pattern comprising one or more of the following characteristics:

(i) at least 20% of the complex-type carbohydrates include a bisecting N-acetylglucosamine; and

(iii) at least 85% of the complex-type carbohydrates include a core fucose.

14. An isolated human cell comprising an expression vector comprising a polynucleotide encoding a human surfactant protein D (SP-D) polypeptide, wherein the cell is selected from the group consisting of NM-H9D8, NM-H9D8-E6Q12, NM-H9D8(8B11), and NM-F9; and

wherein the cell is capable of expressing the SP-D polypeptide having an antennarity number that is within 10% from an antennarity number for a naturally occurring human SP-D, or wherein the an antennarity number is within a range from 190 to 215.

15. The cell of claim 14 , wherein the antennarity number for the expressed SP-D polypeptide and the antennarity number for a naturally occurring human SP-D are within 4% from each other.

16. The cell of claim 14 , wherein the expressed SP-D polypeptide comprises an N-glycosylation profile comprising a peak at 50 minutes having a magnitude within 10% from a magnitude of a peak at 50 minutes in an N-glycosylation profile for a naturally occurring human SP-D.

17. The cell of claim 14 , wherein the cell is a NM-H9D8 cell.

18. The cell of claim 14 , wherein the cell is a NM-H9D8(8B11) cell.

Assignments (3)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Sep 25, 2020
From: ROSENBAUM, JAN SUSAN
To: AIRWAY THERAPEUTICS, LLC
Reel/Frame 053887/0405 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Sep 25, 2020
From: QUAST, FREDERICK GYAPON; KAUP, MATTHIAS; STÖCKL, LARS
To: GLYCOTOPE GMBH
Reel/Frame 053888/0799 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Sep 25, 2020
From: AIRWAY THERAPEUTICS, LLC
To: AIRWAY THERAPEUTICS, INC.
Reel/Frame 053891/0451 →
Continuity (4)
Continuation 16121039 · Sep 4, 2018
Provisional Application 62614758 · Jan 8, 2018
Provisional Application 62554825 · Sep 6, 2017
Related Publication 20200407747A1 · Dec 31, 2020
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