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

Abstract

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

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 614,774, entitled "METHODS AND COMPOSITIONS FOR PREPARING SURFACTANT PROTEIN D (SP-D)," filed January 8, 2018, and U.S. Provisional Application No. 62 / 554,777, entitled "METHODS AND COMPOSITIONS FOR PREPARING SURFACTANT PROTEIN D (SP-D)," filed September 6, 2017, each of which is incorporated by reference in its entirety.

[0002] Sequence Listing Reference This application has been filed with an electronic Sequence Listing, which is provided in a file entitled AIRWY009WOSEQ, approximately 13 Kb in size, created on August 31, 2018. The information in the electronic Sequence Listing is incorporated herein by reference in its entirety.

[0003] Some embodiments of the methods and compositions provided herein relate to the preparation of surfactant protein D (SP-D). Some embodiments involve the expression of human SP-D in certain cell lines and the purification of human SP-D from such cell lines. Some embodiments involve the preparation of certain oligomeric forms of human SP-D. [Background technology]

[0004] Mammalian pulmonary surfactant is a mixture of protein (10%) and lipids (90%), with dipalmitoylphosphatidylcholine being the major lipid component (Zuo YY et al., Biochim Biophys Acta (2008) 1778:1947-77). The main function of pulmonary surfactant is to ensure minimal surface tension in the lungs to prevent collapse during breathing. Furthermore, pulmonary surfactant also participates in host defense by interacting with inhaled pathogens (Clements JA. Am Rev Respir Dis (1977) 115:67-71). Therefore, pulmonary surfactant deficiency is associated with pulmonary diseases such as asthma, bronchiolitis, respiratory distress syndrome (RDS), cystic fibrosis, and pneumonia (Griese M. Eur Respir J (1999) 13:1455-76). Surfactant preparations are indicated for the treatment of RDS, which affects approximately 1.5 million premature infants worldwide each year. Respiratory distress syndrome is a primary pulmonary surfactant deficiency caused by underdeveloped lung structure in premature infants, which leads to respiratory difficulty, impaired gas exchange, and accelerated alveolar collapse (Notter RH. Lung Surfactants. Basic Science and Clinical Applications. New York, NY: Marcel Dekker Inc.). However, current surfactant preparations lack surfactant protein D (SP-D), making treatment more difficult when the lung is infected or when inflammatory or oxidative complications are present. Therefore, successful treatment of complex pulmonary diseases requires the generation of surfactant preparations whose composition matches as closely as possible to that of natural pulmonary surfactant (Robertson B et al., Biochim Biophys Acta (1998) 1408:346-61).

[0005] SP-D plays a role in the pulmonary innate immune system by providing anti-inflammatory and antibacterial activities that address chronic lung diseases such as asthma, cystic fibrosis, and smoking-induced emphysema (Clark H et al., Immunobiology (2002) 205:619-31). Data based on premature newborn lambs suggests that administration of approximately 2-3 mg / kg of recombinant human SP-D in combination with 100 mg / kg of Survanta® (a natural surfactant available in the United States) is more effective in preventing endotoxic shock and reducing ventilation-induced lung inflammation than Survanta® alone (Ikegami M et al., Am J Respir Crit Care Med (2006) 173:1342-7; Sato A et al., Am J Respir Crit Care Med (2010) 181:1098-105).

[0006] Traditionally, SP-D is isolated from the supernatant of bronchoalveolar lavage fluid or amniotic fluid, but most SP-D is lost during purification, partly due to its hydrophilic properties (Dodagatta-Marri E et al., Methods Mol Biol (2014) 100:273-90). The use of native SP-D to supplement pulmonary surfactant preparations can ensure therapeutic efficacy by increasing the number of SP-D binding sites for carbohydrate ligands on the surface of pathogens through higher-order multimerization of endogenous surfactants, achieving a potent bacterial and viral agglutination effect (White M et al., J Immunol (2008) 181:7936-43). The proper oligomerization state is also required for receptor recognition and receptor-mediated signaling for regulation of host immune responses (Yamoze M et al., J Biol Chem (2008) 283:35878-35888), and for maintaining surfactant homeostasis (Zhang L et al., J Biol Chem (2001) 276:19214-19219).

[0007] The use of natural sources for the production of pharmaceutical SP-D is difficult due to low SP-D yields and variable oligomerization states (Strong P et al., J Immunol Methods (1998) 220:139-49). To overcome some of these limitations, recombinant SP-D can be produced in microorganisms or mammalian cell lines, potentially providing a large-scale platform for the production of homogeneous recombinant SP-D preparations. However, it is difficult to express recombinant human SP-D (rhSP-D) in commonly used mammalian cell lines to levels sufficient for commercial activity because the protein is not synthesized efficiently, with yields typically being less than 2 mg of purified protein per liter. Although yields tend to be higher in non-mammalian systems, attempts have been made to express only truncated variants of SP-D in systems such as yeast or bacteria, which have the disadvantage of not producing glycosylated forms of the protein or proteins with human glycosylation patterns (Salgado D et al., Front Immunol (2014) 5:623, doi: 10.3389 / fimmu.2014.00623). Furthermore, to date, it has not been possible to control for the variability in oligomerization states observed in recombinant and native human SP-D. Unless an expression system can reproducibly produce consistently stable levels of rhSP-D with higher-order multimerization states than those observed in native SP-D, the potential exists for reduced efficacy of such preparations. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] U.S. Patent No. 9,051,356 [Patent Document 2] U.S. Patent No. 9,359,427 [Non-patent literature]

[0009] [Non-Patent Document 1] Zuo YY et al., Biochim Biophys Acta (2008) 1778:1947~77 pages [Non-licensed Document 2] Clements JA. Am Rev Respir Dis (1977) 115:67~71 pages [Non-licensed Document 3] Griese M. Eur Respir J (1999) 13: 1455~76 [Non-licensed Document 4] Notter RH. Lung Surfactants. Basic Science and Clinical Applications. New York, NY: Marcel Dekker Inc. [Non-licensed Document 5] Robertson B et al., Biochim Biophys Acta (1998) 1408: 346-61 [Non-licensed Document 6] Clark H et al., Immunobiology (2002) 205: 619-31 [Non-licensed Document 7] Ikegami M et al., Am J Respir Crit Care Med (2006) 173:1342~7 pages [Non-licensed Document 8] Sato A et al., Am J Respir Crit Care Med (2010) 181:1098~105 pages [Non-licensed Document 9] Dodagatta-Marri E et al., Methods Mol Biol (2014) 100:273~90 pages [Non-licensed Document 10] White M et al., J Immunol (2008) 181:7936~43 pages [Non-licensed Document 11] Yamoze M et al., J Biol Chem (2008) 283:35878~35888 pages [Non-licensed Document 12] Zhang L et al., J Biol Chem (2001) 276:19214~19219 pp. [Non-Patent Document 13] Strong P et al., J Immunol Methods (1998) 220:139–49. [Non-Patent Document 14] Salgado D et al., Front Immunol (2014) 5:623, doi: 10.3389 / fimmu.2014.00623 [Non-Patent Document 15] Crouch E. et al. (1994) J Biol Chem, 269:17311~9 [Non-Patent Document 16] Hakansson K et al. Protein Sci (2000) 9:1607~17 [Non-Patent Document 17] Crouch E. Respir Res (2000) 1:93~108 [Non-Patent Document 18] Crouch E. et al. (2006) J Biol Chem, 281:18008~14 [Non-Patent Document 19] R. Arroyo et al., J Mol Biol (2018) 430: pp. 1495–1509 Summary of the Invention [Means for solving the problem]

[0010] Some embodiments of the methods and compositions provided herein include compositions comprising a human surfactant protein D (SP-D) polypeptide, prepared by a method comprising the steps of: (a) introducing a polynucleotide encoding the SP-D polypeptide into human mammalian cells; (b) culturing the cells under conditions in which the SP-D polypeptide is expressed; and (c) isolating the expressed SP-D polypeptide from the cells.

[0011] In some embodiments, the cells are derived from human myeloid leukemia cells.

[0012] In some embodiments, the cell is selected from the group consisting of NM-H9D8, NM-H9D8-E6Q12, and NM-F9. In some embodiments, the cell is a NM-H9D8 cell. In some embodiments, the cell is a NM-H9D8(8B11) cell.

[0013] In some embodiments, the polynucleotide encodes a wild-type SP-D polypeptide leader sequence.

[0014] In some embodiments, the polynucleotide comprises SEQ ID NO:03.

[0015] In some embodiments, the polynucleotide comprises SEQ ID NO:02.

[0016] In some embodiments, the polynucleotide encodes a polypeptide having an amino acid sequence comprising SEQ ID NO:05.

[0017] In some embodiments, the polynucleotide encodes a polypeptide having an amino acid sequence comprising SEQ ID NO:04.

[0018] In some embodiments, the polynucleotide encodes a wild-type T cell receptor (TCR) polypeptide leader sequence.

[0019] In some embodiments, the polynucleotide comprises SEQ ID NO:08.

[0020] In some embodiments, the polynucleotide comprises SEQ ID NO:07.

[0021] In some embodiments, the polynucleotide encodes a polypeptide having an amino acid sequence comprising SEQ ID NO:10.

[0022] In some embodiments, the polynucleotide encodes a polypeptide having an amino acid sequence comprising SEQ ID NO:09.

[0023] In some embodiments, the SP-D polypeptide comprises a residue at a polymorphic position, the residue being selected from the group consisting of Met11 / 31, Thr160 / 180, Ser270 / 290, and Ala286 / 306. In some embodiments, the SP-D polypeptide comprises Met11 / 31. In some embodiments, the SP-D polypeptide comprises Met11 / 31, Thr160 / 180, Ser270 / 290, and Ala286 / 306.

[0024] In some embodiments, the human SP-D polypeptide is SEQ ID NO: 2 1 From 37 5 It contains the amino acid sequence of position .

[0025] Some embodiments also include a population of expressed SP-D polypeptides, each expressed SP-D polypeptide comprising a complex 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 carbohydrates comprise core fucose, (ii) at least 10% of the complex carbohydrates comprise at least one sialic acid residue, (iii) at least 50% of the complex carbohydrates comprise at least a biantennary carbohydrate structure, (iv) at least 10% of the complex carbohydrates comprise bisecting N-acetylglucosamine, (v) less than 10% of the carbohydrates are high mannose structures, and (vi) a detectable amount of α2,6-linked sialic acid residues.

[0026] In some embodiments, the population has a glycosylation pattern comprising one or more of the following characteristics: (i) at least 20% of the complex carbohydrates comprise bisecting N-acetylglucosamine, and (ii) at least 85% of the complex carbohydrates comprise a core fucose.

[0027] In some embodiments, the polynucleotide encodes a dihydrofolate reductase polypeptide. In some embodiments, the step of culturing the cells comprises contacting the cells with an antifolate. In some embodiments, the expression of the SP-D polypeptide is increased by increasing the concentration of the antifolate. In some embodiments, the antifolate comprises methotrexate.

[0028] In some embodiments, the cells are cultured in a perfusion bioreactor.

[0029] In some embodiments, the cells are cultured in continuous culture.

[0030] In some embodiments, culturing the cells comprises maintaining a growth medium having a pH of 7.2, 40% and / or 20% dissolved oxygen, and a temperature of 37° C. In some embodiments, the dissolved oxygen is less than 35%, preferably 30%.

[0031] In some embodiments, the step of isolating the expressed SP-D polypeptide from the cells comprises preparing a cell supernatant from the culture medium containing the cells.

[0032] Some embodiments of the methods and compositions provided herein include a solution comprising a population of recombinant human surfactant protein D (SP-D) polypeptides having oligomeric forms, wherein greater than about 40% of the oligomeric forms comprise dodecamers of SP-D.

[0033] In some embodiments, the human SP-D polypeptide is SEQ ID NO: 2 1 From 37 5 It contains the amino acid sequence of position .

[0034] In some embodiments, the SP-D polypeptide comprises a residue at a polymorphic position, the residue being selected from the group consisting of Met11 / 31, Thr160 / 180, Ser270 / 290, and Ala286 / 306. In some embodiments, the SP-D polypeptide comprises Met11 / 31. In some embodiments, the SP-D polypeptide comprises Met11 / 31, Thr160 / 180, Ser270 / 290, and Ala286 / 306.

[0035] In some embodiments, greater than about 60% of the oligomeric forms comprise SP-D dodecamers. In some embodiments, greater than about 62% of the oligomeric forms comprise SP-D dodecamers. In some embodiments, greater than about 64% of the oligomeric forms comprise SP-D dodecamers.

[0036] In some embodiments, the distribution of oligomeric forms of SP-D is measured using asymmetric flow field-flow fractionation-multi-angle light scattering (AF4-MALS) analysis.

[0037] In some embodiments, each SP-D polypeptide comprises a complex carbohydrate attached at an N-glycosylation site, and the population has a glycosylation pattern comprising the following characteristics: (i) at least 70% of the complex carbohydrates comprise a core fucose, (ii) at least 10% of the complex carbohydrates comprise at least one sialic acid residue, (iii) at least 50% of the complex carbohydrates comprise at least a biantennary carbohydrate structure, (iv) at least 10% of the complex carbohydrates comprise a biantennary N-acetylglucosamine, (v) less than 10% of the carbohydrates are high-mannose structures, and (vi) a detectable amount of α2,6-linked sialic acid residues. In some embodiments, the population has a glycosylation pattern comprising one or more of the following characteristics: (i) at least 20% of the complex carbohydrates comprise a biantennary N-acetylglucosamine, and (ii) at least 85% of the complex carbohydrates comprise a core fucose.

[0038] Some embodiments of the methods and compositions provided herein include compositions comprising a human surfactant protein D (SP-D) polypeptide, prepared by a method comprising the steps of: (a) culturing human mammalian cells comprising an introduced polynucleotide encoding the SP-D polypeptide under conditions in which the SP-D polypeptide is expressed; and (b) isolating the expressed SP-D polypeptide from the cells.

[0039] In some embodiments, the cells are derived from human myeloid leukemia cells.

[0040] In some embodiments, the cells are selected from the group consisting of NM-H9D8, NM-H9D8-E6Q12, and NM-F9.

[0041] In some embodiments, the cells are NM-H9D8 cells.

[0042] In some embodiments, the cells are NM-H9D8 (8B11) cells. [Brief explanation of the drawings]

[0043] [Figure 1] FIG. 1 is a schematic diagram showing the formation of SP-D trimers and the structural features of SP-D trimers. [Figure 2A] FIG. 2A is a map of the expression vector pHBG1Ddhfr_WT_SP-D (7228 bp) containing a polynucleotide encoding human SP-D, a human SP-D leader sequence, and dihydrofolate reductase (DHFR). [Figure 2B] FIG. 2B is a map of the expression vector pHBG1Ddhfr_TCR_SP-D (7231 bp) containing polynucleotides encoding human SP-D, a human T cell receptor (TCR) leader sequence, and dihydrofolate reductase (DHFR). [Figure 2C]FIG. 2C is a map of the expression vector pHBG1Ddhfr_SFTPD (7228 bp) containing a polynucleotide encoding human SP-D, a human SP-D leader sequence, and dihydrofolate reductase (DHFR). [Figure 3] Figure 3 is a bar graph showing the specific production rates of pools cultured at various methotrexate (MTX) concentrations. The cell line pool included "rhSP-D-F9," "rhSP-D-Fuc(-)," and "rhSP-D-H9D8," which are F9 cells, H9D8-E6Q12 cells, and H9D8 cells transfected with a human SP-D expression vector containing the human SP-D leader sequence, respectively, as well as "rhSP-D-TCR-H9D8," which is H9D8 cells transfected with a human SP-D expression vector containing the human TCR leader sequence. [Figure 4] Figure 4 is a series of graphs showing the changes over time in bioreactor culture conditions run on clone H9D8-P1315-2A5, including viable cell concentration (Panel A), glucose concentration (Panel B), cell viability (Panel C), and lactate concentration (Panel D). [Figure 5] FIG. 5 is a photograph of an SDS-PAGE gel stained with Coomassie blue showing proteins at various stages of rhSP-D purification from expressing cells. [Figure 6] Figure 6 is a line graph of a bacterial aggregation assay in which bacteria were treated with various concentrations of rhSP-D purified from clone H9D8-P1315-2A5, showing how SP-D treatment affected bacterial aggregation over time. [Figure 7] FIG. 7 is a line graph showing the inhibitory activity of increasing concentrations of rhSP-D purified from clone H9D8-P1315-2A5 in a TLR4 receptor pathway assay. [Figure 8] FIG. 8 shows chromatograms of fluorescently tagged N-glycans released from purified rhSP-D of different origins and subjected to fluorescence-detected hydrophilic interaction ultrafast chromatography. [Figure 9]FIG. 9 shows chromatograms of fluorescently tagged O-glycans released from different sources of SP-D and subjected to fluorescence-detected hydrophilic interaction ultrafast chromatography. [Figure 10] Figure 10 shows chromatograms of fluorescently tagged N-glycans released from SP-D produced in CHO (Panels A and B) and NM-H9D8(8B11) (Panels C and D) without (Panels A and C) and with (Panels B and D) neuraminidase S treatment, and subjected to fluorescence-detected hydrophilic interaction ultrafast chromatography. DETAILED DESCRIPTION OF THE INVENTION

[0044] Surfactant protein D (SP-D) is a C-type (Ca) surfactant protein containing four domains: a cysteine-linked N-terminal region required for intermolecular disulfide bond formation, a triple-helical collagen region, an α-helical coiled-coil trimerization neck peptide, and a C-terminal calcium-dependent carbohydrate recognition domain (CRD). 2+SP-D is a lectin (protein-dependent) (Crouch E. et al. (1994) J Biol Chem 269:17311-9). Monomers form trimers by folding the collagenous region into a triple helix and assembling a coiled-coil bundle of α-helices in the neck region (Figure 1). These trimers are stabilized by two disulfide bonds in the cysteine-rich N-terminal domain. SP-D trimers have a total molecular weight of 129 kDa, containing three identical 43 kDa polypeptide chains. SP-D trimers can form higher-order oligomerization states that vary in size and conformation. Higher oligomerization states may be important for SP-D function (Hakansson K et al., Protein Sci (2000) 9:1607-17; Crouch E. Respir Res (2000) 1:93-108; Crouch E. et al. (2006) J Biol Chem, 281:18008-14). The assembly of SP-D trimers into higher oligomerization states is susceptible to environmental factors and conditions during purification and storage. The pathways and types of interactions involved in the formation of large SP-D oligomers have not yet been elucidated. Some embodiments of the methods and compositions provided herein relate to the preparation and purification of certain forms of SP-D oligomers.

[0045] Human SP-D produced in mammalian Chinese hamster ovary (CHO) cells has been characterized by atomic force microscopy (AFM) and electrophoresis. Solutions of rhSP-D can contain a diverse population of different SP-D oligomeric forms, including trimers, hexamers, dodecamers, and larger oligomeric species identified as "fuzzy balls" containing more than four trimers. In some embodiments of the present invention, it has been demonstrated that production of SP-D described herein, particularly using the vectors and / or host cells and / or purification methods described herein, results in higher yields of SP-D protein, higher amounts of SP-D dodecamers, and lower amounts of larger oligomeric species compared to production of SP-D in CHO cells. In some embodiments of the present invention, it has been demonstrated that production of SP-D as described herein, particularly using the vectors and / or host cells and / or purification methods described herein, results in a higher yield of SP-D protein, a higher relative amount of SP-D dodecamer, and a lower relative amount of larger oligomeric species compared to production of rhSP-D in CHO cells. For example, yields can be increased by up to about 5-15 fold, the relative amount of SP-D dodecamer in the purified rhSP-D composition as measured by SEC HPLC can be increased by more than about 30%, and the relative amount of larger oligomeric species can be reduced by more than about 30%. Purification of cell culture supernatants through Q-Sepharose and Superdex 75 columns does not alter the ratio of dodecamer to larger oligomeric species (e.g., fuzzy balls). Therefore, the relative amounts of dodecamer and larger oligomeric species in the purified SP-D composition represent their relative amounts in the cell culture supernatant.

[0046] Certain expression vectors and cells In one aspect, an expression vector is provided comprising a polynucleotide encoding a human SP-D polypeptide. Some embodiments involve the preparation of an expression vector comprising a polynucleotide encoding a human SP-D polypeptide. Polymorphisms in the human SP-D polypeptide include ATG (Met) to ACG (Thr) at residue 11, AGT (Ser) to AGC (Ser) at residue 25, ACA (Thr) to GCA (Ala) at residue 160, TCT (Ser) to ACT (Thr) at residue 270, and GCT (Ala) to GCC (Ala) at residue 286, where these positions are relative to their positions in the mature SP-D polypeptide. In some embodiments, the SP-D polypeptide comprises a particular residue at a polymorphic position, where the residues are selected from Met11 / 31, Thr160 / 180, Ser270 / 290, and Ala286 / 306, and the residue positions are relative to their positions in the mature SP-D polypeptide and in the SP-D polypeptide with the leader polypeptide. In some embodiments, the SP-D polypeptide comprises Met11 / 31. In some embodiments, the SP-D polypeptide comprises Met11 / 31, Thr160 / 180, Ser270 / 290, and Ala286 / 306. Examples of such sequences are provided in TABLE 1. In some embodiments, SP-D is encoded by a nucleic acid having at least about 80%, 90%, 95%, 99%, and 100%, or any range between any of the foregoing figures, identity over the entire length of the polynucleotide to a polynucleotide selected from SEQ ID NO:02 and SEQ ID NO:07. In some embodiments, the SP-D polypeptide has at least about 80%, 90%, 95%, 99%, and 100%, or any range between any of the foregoing figures, identity over the entire length of the polynucleotide to a polypeptide selected from SEQ ID NO:04 and SEQ ID NO:09. In some embodiments, the SP-D polypeptide comprises the 2nd amino acid sequence of SEQ ID NO:04. 1 From 37 5 or an amino acid sequence that is at least 80%, at least 90%, at least 95%, or at least 99% identical over the entire length of the reference sequence.

[0047] In some embodiments, the expression vector encodes a leader polypeptide located 5' to the nucleotide sequence encoding the SP-D polypeptide. In some embodiments, the leader sequence is a wild-type T cell receptor (TCR) leader sequence or a wild-type SP-D leader sequence. Examples of such sequences are provided in TABLE 1. In some embodiments, the leader polypeptide is encoded by a nucleic acid having at least about 80%, 90%, 95%, 99%, and 100%, or any range between any of the foregoing figures, identity over the entire length of the polynucleotide to a polynucleotide selected from SEQ ID NO:03 and SEQ ID NO:08. In some embodiments, the leader polypeptide has at least about 80%, 90%, 95%, 99%, and 100%, or any range between any of the foregoing figures, identity over the entire length of the polynucleotide to a polypeptide selected from SEQ ID NO:05 and SEQ ID NO:10.

[0048] In some embodiments, the expression vector contains a selection gene useful for selecting mammalian cells that carry the selection gene. Examples of such genes include those that encode proteins such as dihydrofolate reductase that confer resistance to antifolate compounds such as methotrexate.

[0049] In one aspect, a cell is provided that contains one or more expression vectors comprising a polynucleotide encoding a human SP-D polypeptide. Some embodiments include cells that contain one or more expression vectors described herein. Examples of such cells include mammalian cells that are capable of modifying the expressed SP-D polypeptide with a glycosylation pattern that enhances the activity and / or stability of the expressed SP-D polypeptide. Such cells include immortalized human blood cells, such as cells derived from human myeloid leukemia. Examples of such cells include NM-H9D8 (DSM ACC 2806), NM-H9D8-E6Q12 (DSM ACC 2856), and NM-F9 (DSM ACC 2606), deposited with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany, under the applicable ACC codes. NM-F9 was deposited by Nemod Biotherapeutics GmbH & Co. KG, Robert-Roessle-Str. 10, 13125 Berlin (DE) on August 14, 2003; NM-H9D8 was deposited by Glycotope GmbH, Robert-Roessle-Str. 10, 13125 Berlin (DE) on September 15, 2006; and NM-H9D8-E6Q12 was deposited by Glycotope GmbH, Robert-Roessle-Str. 10, 13125 Berlin (DE) on August 8, 2007. Further examples of useful cell lines can be found in U.S. Patent No. 9,051,356, which is incorporated herein by reference in its entirety. In some embodiments, the cells containing one or more expression vectors comprising a polynucleotide encoding a human SP-D polypeptide are cells of cell line NM-H9D8.

[0050] Certain methods for producing human SP-D In one aspect, methods for producing a human SP-D polypeptide composition are provided. Some embodiments include methods for producing a human SP-D polypeptide composition by (a) introducing a polynucleotide encoding a human SP-D polypeptide into mammalian cells, (b) culturing the cells under conditions in which the SP-D polypeptide is expressed, and (c) isolating the expressed SP-D polypeptide from the cells. Methods for introducing a polynucleotide encoding an SP-D polypeptide into mammalian cells are well known in the art and include electroporation, transfection using cationic lipids, calcium phosphate, DEAE-dextran, or infection with a viral particle such as an adenovirus or retrovirus, or a combination thereof. Some such methods include linearizing an expression vector provided herein and transfecting the linearized vector into cells. In some embodiments, the cells and / or expression vectors described herein are used in methods for producing a human SP-D polypeptide composition. In some embodiments, the SP-D polypeptide is secreted by the mammalian cells. In these embodiments, the expressed SP-D polypeptide can be isolated from the cell culture medium used to culture the cells. In some embodiments, the step of isolating the expressed SP-D polypeptide is carried out as described herein.

[0051] In some embodiments, the cells are derived from human myeloid leukemia cells, such as NM-H9D8, NM-H9D8-E6Q12, and NM-F9 cell lines.

[0052] Some embodiments include a method for selecting cells containing the expression vector. Some such embodiments can include culturing the cells with an antifolate, such as methotrexate. Transfectants can be isolated by methods such as subcloning, and cells expressing SP-D can be readily identified by immunological methods using antibodies against SP-D in combination with methods well known in the art, such as ELISA, Western blot, and dot blot.

[0053] In some embodiments, transfected cells expressing increased levels of SP-D can be selected by increasing the concentration of an antifolate, such as methotrexate, in the culture medium.

[0054] Some embodiments include culturing cells expressing SP-D in a perfusion bioreactor. Some such embodiments can include culturing the cells by continuous fermentation. In the perfusion mode, fresh medium can be continuously supplied, or the cells can be maintained in the fermenter while the cell-free supernatant is removed from the bioreactor. Different techniques can be applied to retain the cells. For example, filtration, centrifugation, or sedimentation can be used. Examples of methods can be found in U.S. Pat. No. 9,359,427, which is incorporated herein by reference in its entirety.

[0055] Certain methods for isolating SP-D from culture media In one aspect, methods for isolating human SP-D polypeptides are provided. Some embodiments include isolating the expressed SP-D polypeptide from the culture medium. In one embodiment, the isolation is by using chromatography. Examples of chromatographic methods include affinity chromatography using affinity materials, such as protein A, protein G, anti-SP-D antibodies, lectin chromatography, antibodies against specific tags or antigens introduced into the SP-D polypeptide, such as a HIS tag or myc tag, or other chromatographic media, such as ion exchange chromatography, hydrophobic interaction chromatography, mixed-mode chromatography, or size exclusion chromatography.

[0056] In some embodiments, a cell supernatant is prepared from a culture medium containing SP-D-expressing cells. The supernatant may be filter-sterilized. In some embodiments, the cell supernatant containing the SP-D polypeptide may be applied to a column, and the resulting eluate may be applied to a second column. In some embodiments, the SP-D polypeptide is isolated using anion exchange chromatography followed by affinity chromatography. In some embodiments, a strong anion exchange chromatography matrix, such as Q-Sepharose, is used for the anion exchange chromatography. In some embodiments, a gel filtration chromatography matrix, such as Superdex 75 matrix, is used for the affinity chromatography. In some embodiments, the cell supernatant is applied to a Q-Sepharose column with an equilibration and running buffer comprising 20 mM Tris, 50 mM NaCl, pH 7.4. SP-D may be eluted from the column using an elution buffer comprising 20 mM Tris, 600 mM NaCl, pH 7.4. In some such embodiments, the Q-Sepharose column eluate contains about 0.2 to about 0.8 mg / ml of SP-D.

[0057] Some embodiments include applying the fraction of the Q-Sepharose column eluate containing SP-D to a second column, such as a Superdex 75 column. In some embodiments, the Q-Sepharose column eluate is diluted with the same volume of 20 mM Tris buffer, pH 7.4, containing 10 mM CaCl2, and applied to a Superdex 75 column with an equilibration and running buffer containing 20 mM Tris, 300 mM NaCl, 5 mM CaCl2, pH 7.4. SP-D may be eluted from the column using an elution buffer containing 20 mM Tris, 10 mM EDTA, 300 mM NaCl, pH 7.4. In some such embodiments, the eluate contains about 0.5 to about 2 mg / mL of SP-D. In some such embodiments, the eluate contains SP-D with a purity of greater than about 90%. Some embodiments also include dialyzing the eluate into 5 mM histidine buffer containing 200 mM NaCl, 1 mM EDTA, pH 7.0 prior to storage and analysis.

[0058] Post-translational modification of SP-D One embodiment includes a human SP-D polypeptide with a specific pattern of post-translational modifications. Some embodiments include compositions comprising a glycosylated, particularly N-glycosylated, human SP-D polypeptide. In some embodiments, the glycosylated human SP-D polypeptide bears a carbohydrate structure at the asparagine corresponding to Asn90 in SEQ ID NO:4 or SEQ ID NO:9. The carbohydrate structure at the N-glycosylation site may comprise a core structure of two N-acetylglucosamine (GlcNAc) residues and three mannose residues, where the first GlcNAc is attached to the polypeptide backbone, the second GlcNAc is attached to the first GlcNAc, the first mannose is attached to the second GlcNAc, and the second and third mannose residues are each attached to the first mannose. Additional monosaccharide units may be attached to this core structure. In some embodiments, at least 60%, particularly at least 70%, at least 75%, at least 80%, at least 85%, or particularly at least 90% of the carbohydrate structures at the N-glycosylation sites of SP-D in the composition are complex carbohydrate structures that contain at least one additional GlcNAc residue attached to a second or third mannose residue but do not contain any additional mannose residues.

[0059] In some embodiments, the human SP-D polypeptide in the composition has the following characteristics at the N-glycosylation site: (i) a relative amount of carbohydrate structures bearing a core fucose of at least 70% of the total amount of complex carbohydrate structures attached to the N-glycosylation site of SP-D in the composition, and / or (ii) a relative amount of carbohydrate structures bearing at least one sialic acid residue of at least 10% of the total amount of complex carbohydrate structures attached to the N-glycosylation site of SP-D in the composition, and / or (iii) a relative amount of at least biantennary carbohydrate structures that is at least 50% of the total amount of complex carbohydrate structures attached to the N-glycosylation sites of SP-D in the composition; and has a glycosylation pattern comprising one or more of:

[0060] In some embodiments, the relative amount of carbohydrate structures bearing core fucose is at least 75%, or at least 80%, of the total amount of complex carbohydrate structures attached to the N-glycosylation site of SP-D in the composition. The core fucose residue is attached to the first GlcNAc residue of the core structure. The "relative amount of carbohydrate structures" according to the present invention refers to a specific percentage or percentage range of carbohydrate structures attached to SP-D in the composition. In particular, the relative amount of carbohydrate structures refers to a specific percentage or percentage range of all carbohydrate structures attached to the SP-D polypeptide chain in the composition. In some embodiments, only carbohydrate structures attached to the N-glycosylation site of SP-D are considered.

[0061] In some embodiments, the relative amount of carbohydrate structures bearing at least one sialic acid residue is at least 15%, at least 20%, or at least 25% of the total amount of complex carbohydrate structures attached to N-glycosylation sites of SP-D in the composition. The relative amount of carbohydrate structures bearing at least one sialic acid residue may range from 10% to 80%, from 15% to 75%, or from 20% to 70%. In some embodiments, the glycosylation pattern of the human SP-D polypeptide in the composition comprises a relative amount of carbohydrate structures bearing two sialic acid residues that is at least 0.5%, e.g., at least 1% or at least 2%, of the total amount of complex carbohydrate structures attached to N-glycosylation sites of SP-D in the composition. The relative amount of carbohydrate structures bearing at least two sialic acid residues may range from 0.5% to 30%, from 1% to 20%, or from 1.5% to 15%. The term "sialic acid" particularly refers to any N- or O-substituted derivative of neuraminic acid. Sialic acid can refer to both 5-N-acetylneuraminic acid and 5-N-glycolylneuraminic acid, but preferably refers only to 5-N-acetylneuraminic acid. Sialic acid, particularly 5-N-acetylneuraminic acid, is preferably linked to the carbohydrate chain via a 2,3- or 2,6-linkage. Preferably, in the glycosylation pattern of SP-D described herein, both 2,3- and 2,6-linked sialic acid are present.

[0062] In some embodiments, the relative amount of at least biantennary carbohydrate structures is at least 60%, or at least 70%, of the total amount of complex carbohydrate structures attached to the N-glycosylation sites of SP-D in the composition. In some embodiments, the glycosylation pattern of the human SP-D polypeptide in the composition comprises a relative amount of at least triantennary carbohydrate structures that is at least 2%, e.g., at least 3%, or at least 4%, of the total amount of complex carbohydrate structures attached to the N-glycosylation sites of SP-D in the composition. A branch is a branch or one or more monosaccharide units attached to a terminal (i.e., second or third) mannose residue of a core structure. In complex carbohydrate structures, the branch generally comprises a GlcNAc residue and may further comprise a galactose residue and, optionally, a sialic acid residue. A biantennary complex carbohydrate structure comprises two branches, i.e., at least a GlcNAc residue is attached to each of the two terminal mannose residues of the core structure. In a triantennary complex carbohydrate structure, one terminal mannose has two branches and the other terminal mannose has one branch. In a tetraantennary complex carbohydrate structure, both terminal mannoses each have two branches. The term "at least biantennary" includes bi-, tri-, and tetraantennary carbohydrate structures, and the term "at least triantennary" includes tri- and tetraantennary carbohydrate structures.

[0063] The A number in glycosylation is a reference number for the branching of glycan structures in a glycosylation pattern. The A number is calculated by multiplying the relative amount of a particular branching by the number of branches and adding the resulting numbers for each branching. Specifically, the relative amount of monoantennary glycans is multiplied by 1, the relative amount of biantennary glycans is multiplied by 2, the relative amount of triantennary glycans is multiplied by 3, and the relative amount of tetraantennary glycans is multiplied by 4. The sum of these numbers results in the A number. In some embodiments, the human SP-D polypeptide in the composition has a glycosylation pattern with an A number of at least 185, e.g., at least 190, at N-glycosylation sites.

[0064] In some embodiments, the human SP-D polypeptide in the composition has the following characteristics at the N-glycosylation site: (i) a relative amount of carbohydrate structures bearing bisecting N-acetylglucosamine (bisGlcNAc) of at least 2%, e.g., at least 5% or at least 8% of the total amount of complex carbohydrate structures attached to N-glycosylation sites of SP-D in the composition; and / or (ii) a relative amount of carbohydrate structures bearing at least one galactose residue of at least 40%, e.g., at least 45% or at least 50% of the total amount of complex carbohydrate structures attached to N-glycosylation sites of SP-D in the composition; and / or (iii) a relative amount of carbohydrate structures bearing at least two galactose residues of at least 15%, e.g., at least 20% or at least 25% of the total amount of complex carbohydrate structures attached to N-glycosylation sites of SP-D in the composition; and / or (iv) a relative amount of carbohydrate structures bearing N-acetylgalactose residues of 30% or less, e.g., 20% or less or 15% or less, of the total amount of complex carbohydrate structures attached to N-glycosylation sites of SP-D in the composition; and / or (v) a relative amount of hybrid carbohydrate structures of 30% or less, e.g., 25% or less, or 20% or less, of the total amount of carbohydrate structures attached to N-glycosylation sites of SP-D in the composition; and / or (vi) a relative amount of high-mannose carbohydrate structures of 25% or less, e.g., 20% or less or 15% or less, of the total amount of carbohydrate structures attached to the N-glycosylation sites of SP-D in the composition; and has a glycosylation pattern comprising one or more of:

[0065] The bisecting N-acetylglucosamine or bisGlcNAc residue is a GlcNAc residue attached to the central (i.e., first) mannose residue of the core carbohydrate structure. In some embodiments, the relative amount of carbohydrate structures bearing bisGlcNAc ranges from 2% to 50%, e.g., 5% to 40%, or 8% to 35%, of the total amount of complex carbohydrate structures attached to the N-glycosylation sites of SP-D in the composition. A "high mannose carbohydrate structure" contains only mannose residues attached to the terminal mannose of the core structure. A "hybrid carbohydrate structure" contains a mannose residue attached to one terminal mannose of the core structure and a branch, as described for a complex carbohydrate structure, attached to the other terminal mannose of the core structure.

[0066] In some embodiments, the population of SP-D polypeptides having complex carbohydrates attached at N-glycosylation sites of SP-D exhibits the following characteristics: (i) at least 20% of the complex carbohydrates contain bi-branched N-acetylglucosamine; (ii) at least 25% of the complex carbohydrates contain at least one sialic acid residue; (iii) at least 85% of the complex carbohydrates contain biantennary carbohydrate structures; (iv) at least 0.5% of the complex carbohydrates contain at least one GalNAc; (v) less than 2% of complex carbohydrates contain three galactoses; and (vi) Less than 2% of the complex carbohydrates contain triantennary carbohydrate structures. The glycosylation pattern may include one or more of:

[0067] In some embodiments, a population of SP-D polypeptides having complex carbohydrates attached at N-glycosylation sites on SP-D can have a glycosylation pattern comprising any of the following characteristics: In some embodiments, at least 15%, 18%, 19%, 20%, 25%, 30%, 35%, 38%, 40%, 45%, or a percentage ranging between any of the foregoing percentages, of the carbohydrate structures attached at the N-glycosylation sites on SP-D in the population comprise biantennary N-acetylglucosamine; In some embodiments, at least 75%, 80%, 82%, 85%, 90%, 95%, or a percentage ranging between any of the foregoing percentages, of the complex carbohydrates attached at the N-glycosylation sites on SP-D in the population comprise biantennary carbohydrate structures. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 15%, or a percentage ranging between any of the foregoing percentages, of the carbohydrate structures attached to the N-glycosylation sites of the SP-D in the population comprise at least one GalNAc. In some embodiments, less than 15%, 10%, 5%, 4%, 3%, 2%, 1%, or a percentage ranging between any of the foregoing percentages, of the carbohydrate structures attached to the N-glycosylation sites of the SP-D in the population comprise three galactose residues. In some embodiments, less than 15%, 13%, 10%, 5%, 4%, 3%, 2%, 1%, or a percentage ranging between any of the foregoing percentages, of the complex carbohydrates attached to the N-glycosylation sites of the SP-D in the population comprise triantennary carbohydrate structures.

[0068] In some embodiments, the human SP-D polypeptide in the composition has the following characteristics at the N-glycosylation site: (i) a relative amount of carbohydrate structures bearing a bisecting GlcNAc of at least 10% of the total amount of complex carbohydrate structures attached to N-glycosylation sites of SP-D in the composition; (ii) a relative amount of high mannose carbohydrate structures that is 10% or less of the total amount of carbohydrate structures attached to the N-glycosylation sites of SP-D in the composition; and (iii) complex carbohydrate structures bearing detectable amounts of α2,6-linked sialic acid residues The glycosylation pattern comprises:

[0069] In a further embodiment, the human SP-D polypeptide in the composition has the following characteristics at the N-glycosylation site: (i) a relative amount of carbohydrate structures bearing a core fucose of at least 85% of the total amount of complex carbohydrate structures attached to the N-glycosylation site of SP-D in the composition; (ii) a relative amount of carbohydrate structures bearing a bisecting GlcNAc of at least 20% of the total amount of complex carbohydrate structures attached to N-glycosylation sites of SP-D in the composition; (iii) a relative amount of high mannose carbohydrate structures that is 10% or less of the total amount of carbohydrate structures attached to the N-glycosylation sites of SP-D in the composition; and (iv) complex carbohydrate structures bearing detectable amounts of α2,6-linked sialic acid residues The glycosylation pattern comprises:

[0070] Identification of oligomeric species of SP-D In one aspect, methods for identifying oligomeric species of human SP-D polypeptides are provided. Some embodiments include methods for identifying oligomeric species of SP-D, such as trimers, dodecamers, and oligomeric structures containing more than four trimers. Such methods may be useful for identifying conditions and ingredients for preparing formulations of SP-D having a particular amount of a particular oligomeric form, e.g., predominantly the dodecamer form. In some embodiments, methods for identifying oligomeric species of human SP-D polypeptides may include performing asymmetric flow field-flow fractionation-multi-angle light scattering (AF4-MALS) analysis on a sample of SP-D. In some embodiments, methods may include performing size-exclusion chromatographic HPLC (SEC HPLC) to identify oligomeric species of human SP-D polypeptides. Examples of SEC HPLC conditions include: UHPLC: Dionex UltiMate 3000; column: TSKgel G6000PWXL, phase: hydroxylated methacrylate, L×ID 30 cm×7.8 mm, 13 μm particle size (#0008024, Tosoh); column oven temperature: 30°C; sampler temperature: 4°C; upper pressure limit: 31 bar; UV detection: 280 nm; eluent: TBS, 10 mM EDTA, pH 7.4 (10×TBS Roti-Stock #1060.1, Carl Roth; EDTA #8040.2, Carl Roth); flow rate: 0.25 mL / min; sample injection: 20 μg or 30 μL fixed volume; integration limits: HOO 25.0-30.0 min, dodecamer 30.0-34.5 min, LOO Examples include 34.5 to 44.0 minutes.

[0071] In some embodiments, a method for identifying SP-D oligomeric species may include performing atomic force microscopy (AFM) on a sample of SP-D, i.e., identifying and / or quantifying SP-D oligomeric species in AFM images. In some such embodiments, the method may include size-separating a mixture of SP-D oligomeric species. Some such methods include contacting the SP-D sample with an anionic detergent, e.g., sodium dodecyl sulfate (SDS), contacting the sample with a cross-linking reagent, e.g., 1% glutardialdehyde (GA), and size-separating the SP-D species, e.g., by performing polyacrylamide gel electrophoresis (PAGE). In some embodiments, the SP-D sample is contacted with the anionic detergent followed by contact with the cross-linking reagent. In other embodiments, the SP-D sample is contacted with the cross-linking reagent followed by contact with the anionic detergent. In some embodiments, the sample is contacted with a solution of about 1% GA. In some embodiments, the sample is contacted with the cross-linking reagent for a period of between about 1 minute and about 30 minutes. In some embodiments, the PAGE is in the presence of sodium dodecyl sulfate (PAGE-SDS). In other embodiments, the PAGE is non-denaturing PAGE. In some embodiments, the PAGE comprises a gradient gel. In some embodiments, the gradient gel is a 4-15% polyacrylamide gradient Tris-glycine gel. In some embodiments, the PAGE is performed in the absence of a reducing agent. In some embodiments, the reducing agent comprises β-mercaptoethanol. Some embodiments also include identifying the SP-D species, for example, by performing a Western blot.

[0072] Certain compositions containing SP-D Some embodiments include a solution comprising a population of rhSP-D polypeptides having a particular distribution of oligomeric forms of SP-D. In some embodiments, the solution can comprise oligomeric forms of SP-D, where greater than about 30%, 40%, 50%, 60%, 61%, 62%, 63%, 64%, 65%, 70%, or any range between the aforementioned figures, of the oligomeric forms comprises SP-D dodecamers. In some embodiments, the distribution of oligomeric forms of SP-D can be measured by a method provided herein, such as asymmetric flow field-flow fractionation-multi-angle light scattering (AF4-MALS) analysis. In some embodiments, the solution of rhSP-D polypeptides is prepared by a method provided herein. In some embodiments, the method for producing a human SP-D polypeptide composition described herein produces a solution comprising a population of rhSP-D polypeptides described herein. In some embodiments, the method for isolating human SP-D polypeptides described herein produces a solution comprising a population of rhSP-D polypeptides described herein. In some embodiments of the methods for producing a human SP-D polypeptide composition described herein, the expressed SP-D polypeptide is predominantly in dodecameric form. In some embodiments of the methods for isolating a human SP-D polypeptide described herein, the isolated SP-D polypeptide is predominantly in dodecameric form. In this regard, "predominantly" particularly refers to a relative amount of at least 30% of all SP-D polypeptides in the composition, e.g., at least 40%, at least 45%, at least 50%, or at least 55% of all SP-D polypeptides in the composition. In some embodiments, "predominantly" refers to a relative amount of more than 50% of all SP-D polypeptides in the composition. [Example]

[0073] Example 1 Construction of SP-D expression vector Two expression vectors were developed for the expression of human SP-D in human mammalian cells. One vector contained the wild-type human SP-D leader / signal sequence, and the other vector contained the human T cell receptor (TCR) leader / signal sequence. The TCR leader sequence was selected for one of the expression vectors because the protein will be expressed in human myeloid leukemia cells, which are expected to secrete proteins with a TCR leader sequence with high efficiency.

[0074] Polynucleotides encoding the human SP-D polypeptide and containing either the wild-type human SP-D leader / signal sequence or the human T cell receptor (TCR) leader / signal sequence were synthesized by GENEART (ThermoFisher Scientific). Each polynucleotide was prepared using the following methods for cloning: Hind III (5' end) and Xba I (3'-end) restriction sites, as well as a Kozak consensus sequence. Each polynucleotide was excised from the GENEART (ThermoFisher Scientific) delivery vector by Hind III / Xba I restriction and ligated into the cloning vector pHBG1Ddhfr (Glycotope GmbH, Germany) to obtain the expression vectors pHBG1Ddhfr_WT_SP-D (7228 bp) and pHBG1Ddhfr_TCR_SP-D (7231 bp). See Figures 2A and 2B. Further examples of expression vectors are shown in Figure 2C. The expression vectors were sequenced and restriction mapped to confirm the correct sequence. Certain sequences are listed in Table 1.

[0075] [Table 1A]

[0076] [Table 1B]

[0077] [Table 1C]

[0078] Example 2 Expression of SP-D in mammalian cell lines Prior to transfection, the expression vector was linearized with PvuI and purified with phenol / chloroform and trichloromethane / chloroform. Cell lines were transfected with 7–8 μg of linearized expression vector using a nucleofection kit (AMAXA NUCLEOFECTOR TECHNOLOGY, Lonza, Cologne, Germany) according to the manufacturer's instructions. The following cell lines were transfected with the expression vector: NM-H9D8 (DSM ACC 2806), NM-H9D8-E6Q12 (DSM ACC 2856), and NM-F9 (DSM ACC 2606).

[0079] Pools of cells expressing SP-D were selected using 25 nM methotrexate (MTX) at increasing concentrations up to 50 nM MTX. To obtain cells with increasing levels of SP-D expression, the MTX concentration was increased stepwise from 100 nM to 200 nM to 400 nM MTX. Productivity of SP-D-producing cells was determined by SP-D-specific ELISA (BioVendor GmbH, Germany, Cat# RD194059101) and / or dot blot analysis according to the manufacturer's instructions using SP-D-specific antibodies (Seven Hills Bioreagents, Cincinnati, OH, Cat# WMAB-2D12A88 and Cat# WMAB-1A10A9). Specific production rate (SPR) was calculated using the following equation:

[0080]

number

[0081]

number

[0082] The doubling time was calculated by the following equation: g=log2×(culture time) / log(final cell number / initial cell number)

[0083] SP-D-expressing clones were isolated from cell pools using ClonePix (Molecular Devices) technology and evaluated for productivity. Selected clones were further subcloned to obtain final clones. Clones with productivity exceeding 100 picograms / cell / day (pcd) were obtained. Figure 3 shows the specific production rates of different SP-D-producing cell pools cultured at various MTX concentrations. The cell pools included H9D8-E6Q12 cells, H9D8 cells, and F9 cells transfected with a human SP-D expression vector containing a human SP-D leader sequence, as well as H9D8 cells transfected with a human SP-D expression vector containing a human TCR leader sequence.

[0084] Example 3 Culturing the SP-D-expressing cell line Cells were cultured in serum-free synthetic gene therapy medium (GTM) (Glycotope GmbH, Germany). For a description of GTM culture medium, see, e.g., U.S. Pat. No. 9,359,427, which is incorporated by reference in its entirety. Perfusion process cultures were initiated in 1x GTM and then changed to 2x GTM. Cells were cultured in T-flasks (25 cm) every 2 to 3 days. 2 1 x 10 in a suspension volume of 3 to 6 mL (TPP GmbH, Germany) 5 From 3 x 10 5 Cells were maintained in exponential growth phase by subculturing to a cell concentration of 0.01 cells / mL and incubated at 37°C, 98% humidity, and 8% CO (Integra Biosciences IBS, Biosafe plus, Switzerland, or Thermo / Heraeus, BBD 6220, Germany). 2 , 12 to 30 mL capacity; 150 cm 2The culture was carried out using a spinner flask (50 to 150 mL) and a spinner flask (100 to 1000 mL, Integra Biosciences IBS, Cellspin, Switzerland).

[0085] Typical culture parameters: 2.0 x 10 cells in medium 5 The cells / mL were inoculated. Continuous operation was enabled by feeding 1x GTM at a perfusion rate of 0.5 V / d, which was increased depending on cell growth and nutrient requirements, up to a maximum perfusion rate of 2 reactor volumes per day. When maximum perfusion was achieved with 1x GTM, the feed medium was replaced with modified 2x GTM. The medium was maintained at pH 7.2 by either adding 0.5 M NaOH or injecting CO2. The dissolved oxygen was set at 40% and the temperature at 37°C. It is also possible to lower the dissolved oxygen to less than 40%, for example, to 20% dissolved oxygen. In the latter case, the dodecamer content may increase slightly compared to the process of culturing under 40% dissolved oxygen. 1 L perfusion bioreactor: Laboratory 1 L-scale cultures were performed in a Sartorius Biostat B-DCU 2L Quad system or a 2L BBI Quad system. Dissolved oxygen and pH were measured by standard electrodes (Mettler Torledo InPro 6800 and Mettler-Torledo 405-DPAS-SC-K8S, respectively, Mettler Torledo, Switzerland). Agitation was performed by a three-blade impeller at a stirring speed of 300 to 400 rpm. Perfusion was performed using a 60 cm PES membrane (0.2 μm pore size and 0.15 m 2 The experiment was carried out using an ATF2 module with a membrane area of ​​0.2 L / min (Spectrum, USA) and a flow rate of 0.9 L / min. For process control, cell concentration and viability were determined by Cedex HiRes (Roche, Switzerland) using the trypan blue exclusion principle. Glucose / lactate and glutamine / glutamate were measured by a YSI2700 or YSI2900 Select biochemical analyzer (Yellow Springs Instruments, USA).

[0086] Figure 4 shows the changes over time in bioreactor culture conditions run on clone H9D8-P1315-2A5, including viable cell concentration (Panel A), glucose concentration (Panel B), cell viability (Panel C), and lactate concentration (Panel D).

[0087] Example 4 Purification of SP-D from mammalian cell lines SP-D was found to be secreted from expressing cells. Cell supernatants from SP-D-producing cells were collected from bioreactor runs or other cultures and purified using a Q-Sepharose FF (GE Healthcare) chromatography run in bind-and-elute mode followed by a Superdex 75 chromatography run (Superdex 75, GE Healthcare) in bind-and-elute mode. Chromatography was performed on a GE FPLC system (Akta Explorer, Akta Avant, Akta Pure).

[0088] Q-Sepharose chromatography: The supernatant was sterile filtered, diluted with an equal volume of 20 mM Tris, 10 mM EDTA, pH 7.4, loaded onto a Q-Sepharose column, and eluted by step elution with 600 mM NaCl. Chromatography was performed using the settings shown in Table 2.

[0089] [Table 2]

[0090] Superdex 75 chromatography: The eluate was diluted with the same volume of 20 mM Tris buffer, pH 7.4, containing 10 mM CaCl, loaded onto a Superdex 75 column, and eluted by step elution with 10 mM EDTA. Chromatography was performed using the settings shown in Table 3.

[0091] [Table 3]

[0092] The Superdex eluate contained SP-D at greater than 90% purity, as determined by non-reducing SDS-PAGE after Coomassie blue staining (Figure 5). In Figure 5, the band above 150 kD contains higher oligomers from SP-D. The eluate from the Superdex column was dialyzed at 4°C against 5 mM histidine pH 7.0 buffer containing 200 mM NaCl and 1 mM EDTA before storage and analysis.

[0093] Example 5 Activity of SP-D in bacterial agglutination assay The activity of SP-D purified from clone H9D8-P1315-2A5 was tested in a bacterial agglutination assay. The bacterial agglutination assay was performed by a method substantially similar to the following: Escherichia coli (ATCC: Y1088) was streaked onto a bacterial agar plate and incubated overnight at 37°C. A single colony was selected and used to inoculate an overnight culture, which was shaken overnight at 37°C. 1 mL of the bacterial culture was pipetted into four 1.5 mL centrifuge tubes and centrifuged at 4,000 rpm for 5 minutes. The supernatant was discarded, and the pellet was resuspended in 1 mL of buffer (150 mM HEPES, 20 mM NaCl, pH 7.4). The tubes were centrifuged at 4,000 rpm for 5 minutes, and the pellet was resuspended in 7 mL of buffer. The absorbance of the bacterial suspension was measured at 700 nm in a spectrophotometer. The bacterial suspension was adjusted to obtain an absorbance ranging from 1.0000 to 1.1000. 1 M CaCl2 was added to the suspension to obtain a final concentration of 5 mM CaCl2. Dilutions of rhSP-D in placebo buffer (each dilution in a total volume of 15 μl) were made at the following concentrations: 5, 1, 0.5, 0.25, 0.1, and 0 μg / ml, and each was added to a cuvette containing 20 μL of HEPES-NaCl buffer. 600 μL of bacterial suspension was then added to the cuvette, and the absorbance was measured at 700 nm for each cuvette every 2.5 minutes for a total of 120 minutes.

[0094] In the agglutination assay, active SP-D aggregates bacterial cells, decreasing the absorbance / increasing the transmittance of the bacterial suspension. Figure 6 shows that purified rhSP-D from clone H9D8-P1315-2A5 was determined to be active in the bacterial agglutination assay. The experiment was repeated two more times with similar results.

[0095] Additionally, recombinantly expressed human SP-D in additional clones of H9D8 generated as described in Example 2 above was analyzed for its activity. SP-D from all tested final selected clones had similar high activity. One exemplary isolated clone, NM-H9D8(8B11), was also used in subsequent analyses. NM-H9D8(8B11) was deposited by Airway Therapeutics LLC, Cincinnati, Ohio, USA, with DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany, on September 4, 2018, under the accession number "AT100-rhSP-D-H9D8-P20011-8B11," and the deposited clone can be easily identified therefrom and obtained under the accession number.

[0096] Example 6 Activity of SP-D in the TLR4 inhibition assay Oligomeric forms of SP-D inhibit lipopolysaccharide (LPS)-induced inflammatory cell responses by preventing LPS from binding to / activating Toll-like receptor 4 (TLR4). See, e.g., Yamazoe M. et al. (2008) J. Biological Chem. 283:35878-35888, which is incorporated by reference in its entirety.

[0097] The activity of rhSP-D purified from clone H9D8-P1315-2A5 in inhibiting LPS-induced activation of the TLR4 pathway was tested. HEK-Blue™ hTLR4 cells (InvivoGen, San Diego, CA, USA) were plated in 384-well plates at a density of approximately 20,000 cells / well and incubated with various concentrations of SP-D for 2 hours at 37°C and 5% CO2. EC 80 A concentration of LPS (E. coli O26:B6, L5543 Sigma-Aldrich) was added to each well, and the cells were incubated for an additional 22 hours at 37°C and 5% CO2. TLR4 activity was measured by detaching the cells from the wells, washing the suspended cells, resuspending the cells in PBS, and gently pipetting to remove any clumps. The washed cells were plated in 20e10 wells of a 384-well plate containing HEK blue detection medium (InvivoGen, San Diego, CA, USA) made with endotoxin-free water containing 5 mM CaCl2 and 1% (v / v) BSA. 3 The cells were plated at a density of 100 cells / well. The cells were incubated at 37°C in 5% CO2 for 24 hours, and TLR4 activity was determined by measuring the activity of the secreted embryonic alkaline phosphatase (SEAP) reporter gene using a spectrophotometer at 655 nm. The IC of SP-D was 50 The IC values ​​were calculated using nonlinear regression analysis by fitting the data to a four-parameter logistic equation using XLfit from idbs (www.idbs.com). Figure 7 shows that purified SP-D from clone H9D8-P1315-2A5 has the activity to inhibit the activation of the TLR4 pathway by LPS, and the IC 50 The IC was determined to be 0.00294 mg / ml, and SP-D was confirmed to be the active oligomeric form for such activity. 50 Only the logarithm of the values ​​is normally distributed, so for the purpose of averaging numbers from a series of experiments, -Log 10 (I C 50 ) defined as pIC 50The experiment was repeated two more times, with similar results, resulting in a mean pIC of 2.33 ± 0.10 mg / ml (N = 3). 50 yielding an average IC of 0.00468 mg / ml 50 The activity of human SP-D expressed recombinantly in additional clones of H9D8, generated as described in Example 2 above, was also analyzed for its activity in the TLR-4 assay. SP-D from all final selected clones tested had similar high activity. One exemplary isolated clone is NM-H9D8(8B11), which was also used in subsequent analyses.

[0098] Example 7 Stability of SP-D from various sources The stability of rhSP-D from various sources was determined. Sources included rhSP-D expressed with a wild-type SP-D leader polypeptide in H9D8 cells ("rhSP-D:WT") and rhSP-D expressed with a TCR leader polypeptide in H9D8 cells ("rhSP-D:TCR"). Solutions of rhSP-D:WT or rhSP-D:TCR in various buffers (Buffers 1, 2, 3, or 4) were incubated at 5°C for several weeks. The stability of rhSP-D:WT or rhSP-D:TCR in the various buffers was determined by measuring the relative distribution of rhSP-D oligomeric forms, including rhSP-D trimers / hexamers, dodecamers, higher-order oligomers known as "fuzzy balls," and very high-order oligomers / aggregates. The relative distribution of rhSP-D oligomeric forms was determined by asymmetric flow field-flow fractionation (AF4)-multi-angle light scattering (AF4-MALS) analysis using essentially the same method as provided in Example 8. Results were averaged from triplicate determinations + / - standard deviation. The results are summarized in TABLE 4.

[0099] [Table 4A]

[0100] [Table 4B]

[0101] Table 4 illustrates the differences in the relative stability of various oligomeric forms in different solutions containing rhSP-D:WT and rhSP-D:TCR. For example, with respect to very high-order oligomeric forms of SP-D, solutions of rhSP-D:WT generally had a lower percentage of such oligomers than the corresponding solutions of rhSP-D:TCR. In addition, the percentage of very high-order oligomers in solutions of rhSP-D:WT did not increase substantially compared with the corresponding solutions of rhSP-D:TCR, at least between weeks 0 and 2. With respect to the dodecamer oligomeric forms of rhSP-D, the percentage of such oligomers in solutions of rhSP-D:WT was generally stable between weeks 0 and 8; in contrast, the percentage of such oligomers in solutions of rhSP-D:TCR decreased in the corresponding solutions between weeks 0 and 8.

[0102] These differences between the rhSP-D:WT and rhSP-D:TCR solutions were noteworthy because both SP-D polypeptides have the same amino acid sequence and are each produced by H9D8 cells. rhSP-D:WT was initially expressed with a leader / signal polypeptide identical to that of the wild-type human SP-D protein, and rhSP-D:TCR was initially expressed with a leader / signal polypeptide identical to that of the human TCR protein. It is possible that each leader / signal sequence was cleaved from its corresponding protein immediately after or during translocation. This finding may be particularly advantageous for the generation and development of stable solutions of human SP-D for the treatment of various pulmonary disorders, particularly solutions containing the more active dodecamer oligomeric form of SP-D.

[0103] Example 8 AF4-MALS analysis Asymmetric flow field-flow fractionation-multiangle light scattering (AF4-MALS) analysis was used to determine the relative distribution of different oligomeric forms of SP-D in solution. AF4-MALS is a separation technique related to field-flow fractionation (FFF). Unlike FFF, AF4-MALS contains a single permeable wall, so that crossflow is driven solely by the carrier liquid. Crossflow is induced by the carrier liquid constantly exiting through a semipermeable wall at the bottom of the channel.

[0104] Samples were analyzed using an AF4-MALS system (Eclipse Dual Tec, Wyatt Technology Corp., Santa Barbara, CA) followed by UV (Ultimate 3000 variable wavelength detector, Dionex Corporation, Sunnyvale, CA) and MALS analysis (Dawn Heleos II detector, Wyatt Technology Corp., Santa Barbara, CA). A Dionex Ultimate 3000 HPLC system (Dionex Corporation, Sunnyvale, CA) was used to inject samples and deliver the mobile phase to the AF4 system. The AF4 configuration used a short channel with a 350 μm thick spacer (Wyatt Technology Corp., Santa Barbara, CA). Data analysis and calculations were performed using Chromeleon (Dionex Corporation, Sunnyvale, CA) and Astra (Wyatt Technology Corp., Santa Barbara, CA) software. Samples included purified rhSP-D from H9D8 or F9 cells transfected with an expression vector encoding rhSP-D and a wild-type SP-D leader polypeptide (pHBG1Ddhfr_WT_SP-D), and H9D8 cells transfected with an expression vector encoding rhSP-D and a wild-type TCR leader polypeptide (pHBG1Ddhfr_TCR_SP-D). The samples used are listed in Table 5. The parameters of the AF4-MALS system for rhSP-D are shown in Table 6.

[0105] [Table 5]

[0106] [Table 6]

[0107] Data collected using AF4-MALS were analyzed using UV and multi-angle light scattering detectors to determine the absolute molar mass and size of SP-D at specific times during elution. The size-to-mass ratio indicated the shape of SP-D. From the size-to-mass ratio, it was determined that SP-D molecules had a linear or rod-like shape in the early stages of elution (0–34 min). For the rod model calculations, the software assumed that the thickness of rod-like particles was insignificant compared to their length (0.0 nm). If the thickness was significant, the thickness in nm or approximate thickness was used. Rod thickness was estimated from atomic force microscopy (AFM) data, and the rod length was determined to be consistent with the AFM measurement of 136 ± 8.1 nm (R. Arroyo et al., J Mol Biol (2018) 430: 1495–1509). The later stages of SP-D elution (34–45 min) indicated that a more compact structure was observed. A second-order Debye model was utilized for the analysis of these stages of elution. The second-order Debye model provided better results over a wider range of molar masses, including very large molar masses (greater than about 10e6 Daltons or about 50 nm RMS radius). The molecular weight of the dodecamer oligomeric form of SP-D was determined to be 520.09 + / - 4.61 kDa (N = 72 determinations).

[0108] The first peak in the elution profile (Peak 1) contained SP-D trimers and hexamers based on mass calculations according to the stick model. The second peak in the elution profile (Peak 2) contained SP-D dodecamers. The third peak in the elution profile (Peak 3) contained an intermediate species between SP-D dodecamers and SP-D "fuzzy balls" based on the intermediate MW determined by the stick model. The fourth peak in the elution profile (Peak 4) contained heterogeneous aggregates of SP-D oligomers with a constant RMS radius of approximately 70 nm, consistent with that observed by AFM measurements for fuzzy ball species. The RMS radius increased beyond 36 minutes in the elution profile, indicating aggregate species.

[0109] Example 9 N-glycan analysis The N-glycosylation patterns of SP-D produced in NM-H9D8 cells (rhSP-D) and SP-D obtained from human amniotic fluid (hSP-D) were compared. Purified SP-D protein was denatured and reduced. N-glycans were released by the action of N-glycanase F. The free N-glycans were tagged with a fluorophore at the reducing end, followed by a purification step using solid-phase extraction. The purified mixture of fluorescently tagged N-glycans was subjected to hydrophilic interaction ultra-high-performance chromatography with fluorescence detection (HILIC-UPLC-FLD) coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-Q-TOF MS / MS). Glycans were quantified by fluorescence peak area and identified by molecular mass using fragment analysis.

[0110] The fluorescence traces showed consistent retention time ranges for all N-glycans. Due to consistent signals across all samples, reliable structural assignments were performed by MS / MS experiments. The glycosylation patterns of the compared SP-D proteins are shown in Table 7.

[0111] [Table 7]

[0112] From the different amounts of branching, the A number can be calculated as a measure of overall branching using the formula: 1 × percentage of monoantennary glycans + 2 × percentage of biantennary glycans + 3 × percentage of triantennary glycans + 4 × percentage of tetraantennary glycans = A number. The A numbers of rhSP-D and hSP-D are very similar, with rhSP-D having an A number of 200 and hSP-D having an A number of 208.

[0113] In some embodiments, the N-glycosylation profiles of hSP-D and rhSP-D were similar. For example, the percentage of carbohydrate structures containing glycans with three sialic acids, monoantennary glycans, tetraantennary glycans, hybrid glycans, or high-mannose glycans was the same for both hSP-D and rhSP-D. However, in some embodiments, the N-glycosylation profiles of hSP-D and rhSP-D were not similar. For example, the percentage of carbohydrate structures containing glycans with biantennary N-acetylglucosamine, glycans with one sialic acid, glycans with three galactoses, triantennary glycans, or glycans with at least one GalNAc differed between hSP-D and rhSP-D.

[0114] Further analysis compared the glycoprofiles of recombinant human SP-D (rhSP-D) produced in different clones of NM-H9D8 cells, including two different purified batches from the NM-H9D8 (8B11) clone, recombinant human SP-D produced in different clones of CHO cells (CHO-SP-D), and native SP-D (hSP-D) obtained from human amniotic fluid. As shown in Figure 8, the N-glycosylation profiles of rhSP-D and hSP-D are highly comparable, whereas CHO-SP-D exhibits significant differences due to its production in a nonhuman cell line. Figure 9 further demonstrates that the O-glycosylation profiles of NM-H9D8-derived rhSP-D and CHO-derived CHO-SP-D are also significantly different. The O-glycosylation of SP-D obtained from human amniotic fluid could not be determined due to the large amount of protein required for this analysis. The N-glycosylation profile includes all glycan structures attached to asparagine residues of the SP-D polypeptide chain, whereas the O-glycosylation profile shows glycan structures attached to serine, threonine, hydroxy-lysine, and hydroxy-proline residues. In conclusion, rhSP-D produced in clones of NM-H9D8 cells has a human glycosylation pattern that closely resembles the glycosylation of naturally occurring hSP-D.

[0115] In addition, we analyzed the linkage of sialic acid (N-acetylneuraminic acid, NANA) in the glycan structure of SP-D. NANA can generally be linked to terminal galactose residues in either the α2,3 or α2,6 conformation. A mixture of α2,3- and α2,6-linked NANA is found in human glycosylation, whereas hamster cells such as CHO do not produce α2,6-linked NANA. CHO and NM-H9D8-produced human SP-D were purified, denatured, and reduced. N-glycans were released during incubation with N-glycanase F. The released N-glycans were labeled with a fluorophore (RapiFluor, Waters), followed by a purification step using HILIC solid-phase extraction. For neuraminidase treatment, purified N-glycans were digested with neuraminidase S (NEB) for 1 hour at 37°C. Neuraminidase S specifically removes α2,3-linked NANA but does not cleave α2,6-linked NANA. The enzyme was removed by repeated HILIC solid-phase extraction. An I-class system (Waters) with fluorescence detection was used for HILIC-UPLC. The purified fluorescently tagged N-glycan mixture was separated on an Acquity UPLC BEH Glycan column (150 x 2.1 mm, 1.7 u, Waters) at a flow rate of 0.5 mL / min and 60 °C. 100% acetonitrile (A) and 100 mM ammonium formate, pH 4.5, were used as the eluent system, with a gradient from 22% B to 44% B over 82 min. The fluorescence wavelength setting was λ ex is 265 nm, λ em The chromatogram was at 425 nm. An attached Bruker Impact HD ESI-Q-TOF-MS (MS) was used in positive ion mode for N-glycan identification. N-glycans were identified according to molecular mass in combination with fragment analysis.

[0116] Analysis revealed that SP-D from CHO cells exhibited a heterogeneous N-glycan profile between 20 and 70 minutes of RT. In addition to three major peaks containing biantennary N-glycans with zero (S0), one (S1), and two (S2) NANA units, more highly branched structures with up to four NANA units were detected between 53 and 70 minutes. The biantennary S1 and S2 structures, as well as the majority of more highly branched structures at RTs from 56 minutes onward, were affected by neuraminidase S treatment, demonstrating the presence of 2,3-linked NANA units. Overall sialylation was significantly reduced, indicating the presence of primarily α2,3-linked NANA units in CHO-produced SP-D (see Figure 10, panels A and B). In SP-D from NM-H9D8 (8B11) cells, only slight changes in the N-glycan profile were detected after neuraminidase treatment. The monosialylated peak at RT 50 min was unaffected by neuraminidase S treatment. In SP-D from NM-H9D8, only one small peak shift containing S2 N-glycans was observed at RT 54 min. Overall sialylation was only slightly reduced by neuraminidase S, indicating that SP-D produced in NM-H9D8(8B11) contained primarily α2,6-linked NANA and only a small amount of α2,3-linked NANA (see Figure 10, panels C and D).

[0117] The term "comprising," as used herein, is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.

[0118] The above description discloses several methods and materials of the present invention. The invention is susceptible to modifications of the methods and materials, and to changes in the manufacturing methods and equipment. Such modifications will become apparent to those skilled in the art from consideration of this disclosure or practice of the invention disclosed herein. It is therefore intended that the present invention not be limited to the specific embodiments disclosed herein, but rather to cover all modifications and alterations falling within the true scope and spirit of the invention.

[0119] All references cited herein, including, but not limited to, published and unpublished applications, patents, and literature references, are incorporated by reference in their entirety and are hereby made a part of this specification. To the extent that the publications, patents, or patent applications incorporated by reference conflict with the disclosure contained herein, it is intended that the present specification supersede and / or take precedence over any such conflicting material.

Claims

1. A solution comprising a population of recombinant human surfactant protein D (rhSP-D) polypeptides having oligomeric forms, wherein greater than about 40% of the oligomeric forms comprise a dodecamer of rhSP-D, and wherein the rhSP-D polypeptides and naturally occurring human SP-D each have the same percentage of carbohydrate structures comprising (i) a glycan having three sialic acids, (ii) a monoantennary glycan, (iii) a tetraantennary glycan, (iv) a hybrid glycan, or (v) a high-mannose glycan; the rhSP-D polypeptide has an A number in the range of 190 to 208; solution.

2. 10. The solution of claim 1, further comprising a histidine buffer.

3. The solution of claim 1, wherein the rhSP-D polypeptide comprises the amino acid sequence from positions 21 to 375 of SEQ ID NO:

04.

4. 2. The solution of claim 1, wherein the rhSP-D polypeptide comprises a residue at a polymorphic position, the residue being selected from the group consisting of Met11 / 31, Thr160 / 180, Ser270 / 290, and Ala286 / 306.

5. The solution of claim 1, wherein the rhSP-D polypeptide comprises Met11 / 31.

6. 2. The solution of claim 1, wherein the rhSP-D polypeptide comprises Met11 / 31, Thr160 / 180, Ser270 / 290, and Ala286 / 306.

7. 2. The solution of claim 1, wherein greater than about 60% of the oligomeric forms comprise a dodecamer of rhSP-D.

8. 2. The solution of claim 1, wherein greater than about 62% of the oligomeric forms comprise a dodecamer of rhSP-D.

9. 2. The solution of claim 1, wherein greater than about 64% of the oligomeric forms comprise a dodecamer of rhSP-D.

10. 2. The solution of claim 1, wherein the distribution of oligomeric forms of the rhSP-D polypeptide is measured using asymmetric flow field-flow fractionation-multi-angle light scattering (AF4-MALS) analysis.

11. wherein each rhSP-D polypeptide comprises a complex carbohydrate attached at an N-glycosylation site, and the population has at least one of the following characteristics: (i) at least 70% of the complex carbohydrates contain core fucose; (ii) at least 10% of the complex carbohydrates contain at least one sialic acid residue; (iii) at least 50% of the complex carbohydrates contain at least a biantennary carbohydrate structure; (iv) at least 10% of the complex carbohydrates contain bi-branched N-acetylglucosamine; (v) less than 10% of the carbohydrates are high mannose structures; and (vi) a detectable amount of α2,6-linked sialic acid residues 2. The solution of claim 1, having a glycosylation pattern comprising:

12. The population has the following characteristics: (i) at least 20% of the complex carbohydrates contain bi-branched N-acetylglucosamine; and (ii) at least 85% of the complex carbohydrates contain core fucose; 12. The solution of claim 11, having a glycosylation pattern comprising one or more of:

13. said population of rhSP-D polypeptides comprising: (a) culturing human myeloid leukemia cells containing an introduced polynucleotide encoding an SP-D polypeptide under conditions in which the SP-D polypeptide is expressed, wherein the cells are selected from the group consisting of NM-H9D8, NM-H9D8-E6Q12, NM-H9D8(8B11), and NM-F9; and (b) isolating the expressed rhSP-D polypeptide from the cells.

10. The solution of claim 1, prepared by a method comprising:

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