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WO 2013/102674 A2 11 July 2013 (11.07.2013) W P O P C T

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WO 2013/102674 A2 11 July 2013 (11.07.2013) W P O P C T (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2013/102674 A2 11 July 2013 (11.07.2013) W P O P C T (51) International Patent Classification: (74) Agent: CABINET PLASSERAUD; 235 cours Lafayette, C12N 1/15 (2006.01) F-69006 Lyon (FR). (21) International Application Number: (81) Designated States (unless otherwise indicated, for every PCT/EP20 13/050 126 kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, (22) Date: International Filing BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, 4 January 2013 (04.01 .2013) DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (25) Filing Language: English HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, (26) Publication Language: English ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, (30) Priority Data: NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, 61/583,559 5 January 2012 (05.01 .2012) US RW, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, (71) Applicants: NOVARTIS INTERNATIONAL PHAR¬ ZM, ZW. MACEUTICAL LTD.; 13 1 Front Street, Hamilton (BM). GLYKOS FINLAND OY [FI/FI]; Viikinkaari 6, FI- (84) Designated States (unless otherwise indicated, for every 00790 Helsinki (FI). kind of regional protection available): ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, (72) Inventors: LANDOWSKI, Christopher; Lohkopellontie UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, 3 A 15, FI-00650 Helsinki (FI). HUUSKONEN, Anne; TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, Mechelininkatu 47 A 29, FI-00250 Helsinki (FI). SAAR- EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, INEN, Juhani; Eljaksentie 3, FI-00370 Helsinki (FI). MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, WESTERHOLM-PARVINEN, Ann; Kuninkaantie 20 A, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, FI-02400 Kirkkonummi (FI). KANERVA, Anne; Riihikal- ML, MR, NE, SN, TD, TG). lionkuja 8, FI-00890 Helsinki (FI). NATUNEN, Jari; Published: Oolannintie 10 E 18, FI-01520 Vantaa (FI). HANNINEN, Anna-Liisa; Karhusuontie 74 A 4, FI-00780 Helsinki (FI). — without international search report and to be republished SALOVUORI, Noora; Laivalahden puistotie 7 A 11, FI- upon receipt of that report (Rule 48.2(g)) 00810 Helsinki (FI). PENTTILA, Merja; Purjeen- — with sequence listing part of description (Rule 5.2(a)) tekijankuja 3 A 1, FI-00210 Helsinki (FI). SALOHEIMO, Markku; Kyntajantie 18b, FI-00390 Helsinki (FI). (54) Title: PROTEASE DEFICIENT FILAMENTOUS FUNGAL CELLS AND METHODS OF USE THEREOF (57) Abstract: The present disclosure relates to compositions and methods useful for the production of heterologous proteins in fi la mentous fungal cells. PROTEASE DEFICIENT FILAMENTOUS FUNGAL CELLS AND METHODS OF USE THEREOF FIELD OF THE INVENTION [0001] The present disclosure relates to compositions and methods useful for the production of heterologous proteins in filamentous fungal cells. BACKGROUND [0002] Posttranslational modification of eukaryotic proteins, particularly therapeutic proteins such as immunoglobulins, is often necessary for proper protein folding and function. Because standard prokaryotic expression systems lack the proper machinery necessary for such modifications, alternative expression systems have to be used in production of these therapeutic proteins. Even where eukaryotic proteins do not have posttranslational modifications, prokaryotic expression systems often lack necessary chaperone proteins required for proper folding. Yeast and fungi are attractive options for expressing proteins as they can be easily grown at a large scale in simple media, which allows low production costs, and yeast and fungi have posttranslational machinery and chaperones that perform similar functions as found in mammalian cells. Moreover, tools are available to manipulate the relatively simple genetic makeup of yeast and fungal cells as well as more complex eukaryotic cells such as mammalian or insect cells (De Pourcq et al., Appl Microbiol Biotechnol, 87(5): 1617-3 1). Despite these advantages, many therapeutic proteins are still being produced in mammalian cells, which produce therapeutic proteins with posttranslational modifications most resembling the native human proteins, whereas the posttranslational modifications naturally produced by yeast and fungi often differ from that found in mammalian cells. [0003] To address this deficiency, new strains of yeast and fungi are being developed that produce posttranslational modifications that more closely resemble those found in native human proteins. Thus, there has been renewed interest in using yeast and fungal cells to express more complex proteins. However, due to the industry's focus on mammalian cell culture technology for such a long time, the fungal cell expression systems such as Trichoderma are not as well established as mammalian cell culture and therefore suffer from drawbacks when expressing mammalian proteins. [0004] Thus, a need remains in the art for improved filamentous fungal cells, such as Trichoderma fungus cells, that can stably produce heterologous proteins, such as immunoglobulins, preferably at high levels of expression. SUMMARY [0005] Described herein are compositions including filamentous fungal cells, such as Trichoderma fungal cells having reduced or no detectable activity of at least three proteases, and having a recombinant polynucleotide encoding a heterologous polypeptide that is produced at increased levels. Further described herein are methods of improving heterologous polypeptide stability and methods of making heterologous polypeptides in which the proteases do not have the reduced activity. [0006] Thus one aspect includes filamentous fungal cells having reduced or no detectable activity of at least three proteases, where the cell further contains a recombinant polynucleotide encoding a heterologous polypeptide produced at a level of at least 2-fold higher than the production level of the polypeptide in a corresponding parental filamentous fungal cell in which the proteases do not have the reduced activity. In certain embodiments, when the cell is an Aspergillus cell, the total protease activity is reduced to 50% or less of the total protease activity of the corresponding parental Aspergillus cell in which the protease do not have reduced activity. In other embodiments, the total protease activity of the filamentous fungal cell is reduced to 49% or less, 31% or less, of the total protease activity of the corresponding parental filamentous fungal cell in which the proteases do not have the reduced activity. [0007] In certain embodiments, the expression level of the at least three proteases is reduced or eliminated. In certain embodiments, genes encoding the three proteases each comprise a mutation that reduces or eliminates the corresponding protease activity. In certain embodiments that may be combined with the preceding embodiments, the three protease encoding genes are pepl, tspl, and slpl. In other embodiments, the three protease encoding genes are gapl, slpl, and pepl. [0008] In certain embodiments, the fungal cells have reduced or no detectable activity of four endogenous proteases; genes encoding the four proteases each comprise a mutation that reduces or eliminates the corresponding protease activity. In certain embodiments that may be combined with the preceding embodiments, the four protease encoding genes are pepl, tspl, slpl, and gapl. [0009] In certain embodiments that may be combined with the preceding embodiments, the three or four protease encoding genes are selected from pepl ,pep2, pep3, pep4, pep5, pep8, pepll, pep!2, tspl, slpl, slp2, slp3, slp7, gapl, and gap2. In certain embodiments that may be combined with the preceding embodiments, the three or four protease encoding genes are selected from pepl ,pep3, pep4, tspl, slpl, slp2, gapl, and gap2. In certain embodiments, the three or four protease encoding genes are selected from pepl, pep2, pep3, pep4, pep5, gapl, gap2, slpl, slp2, and tspl. [0010] In other embodiments, the fungal cells have reduced or no detectable activity of five endogenous proteases; genes encoding the five proteases each comprise a mutation that reduces or eliminates the corresponding protease activity. In certain embodiments that may be combined with the preceding embodiments, the five protease encoding genes are pepl, tspl, slpl, gapl, and pep4. In other embodiments, the five protease encoding genes are pepl, tspl, slpl, gapl, and gap2. [0011] In certain embodiments, the fungal cells have reduced or no detectable activity of six endogenous proteases; genes encoding the six proteases each comprise a mutation that reduces or eliminates the corresponding protease activity. In certain embodiments, the cell has six protease encoding genes, each of which comprise a mutation that reduces or eliminates the corresponding protease activity, and the six protease encoding genes are pepl, tspl, slpl, gapl, gap2, and pep4. [0012] In certain embodiments that may be combined with the preceding embodiments, the fungal cells have three to six proteases having reduced or no detectable activity each of the three to six proteases selected from pepl, pep2, pep3, pep4, pep5, tspl, slpl, slp2, slp3, gapl, and gap2. [0013] In certain embodiments that may be combined with the preceding embodiments,
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