BETA-1,4 GALACTOSYLATION OF PROTEINS

Abstract

The present invention relates to a cell, wherein the cell is modified to: reduce O-GalNAc galactosylation activity in the cell by reduction of functional COSMC molecular chaperone in the cell and/or by reduction of functional T-synthase in the cell; and overexpress β1,4-galactosyltransferase in the cell; and associated methods, kits and uses.

Claims

1. A cell, wherein the cell is modified to: reduce O-GalNAc galactosylation activity in the cell by reduction of functional COSMC molecular chaperone in the cell and/or by reduction of functional T-synthase in the cell; and overexpress β1,4-galactosyltransferase in the cell.

2. The cell according to claim 1, wherein the modification comprises a knockout of the COSMC molecular chaperone or wherein translation from the COSMC molecular chaperone gene is supressed.

3. The cell according to claim 1, wherein the modification comprises a knockout of functional T-synthase in the cell or wherein translation from the T-synthase gene is supressed.

4. The cell according to claim 1, wherein the cell is transformed with nucleic acid encoding β1,4-galactosyltransferase.

5. The cell according to claim 1, wherein the β1,4-galactosyltransferase comprises human β4GalT1.

6. The cell according to claim 1, wherein the cell is selected from CHO, HEK, NS0, SP2/0, PER.C6, Sf9, VERY, BH, HeLa, COS, MDCK, 293, 293T, 3T3, WI38, BT483, Hs578T, HTB2, BT20, T47D, CRL7030 and Hs578Bst.

7. The cell according to claim 1, wherein the cell is modified to express a polypeptide product, wherein the polypeptide product is a heterologous polypeptide.

8. (canceled)

9. The cell according to claim 1, wherein the cell is modified to express a polypeptide product, wherein the polypeptide product comprises one or more, or all the peptides of a therapeutic selected from the group comprising [fam-]trastuzumab deruxtecan, Leronlimab, Narsoplimab, REGNEB3, Sacituzumab govitecan, Tafasitamab, Inebilizumab, Satralizumab, Eptinezumab, Isatuximab, Teprotumumab, Crizanlizumab, Enfortumab vedotin, Polatuzumab vedotin, Risankizumab, Romosozumab, Burosumab, Cemiplimab, Emapalumab, emapalumab-lzsg, Erenumab, Fremanezumab, Galcanezumab, Gemtuzumab ozogamicin, Ibalizumab, ibalizumab-uiyk, Lanadelumab, Mogamulizumab, Ravulizumab (ALXN1210), Tildrakizumab, Avelumab, Benralizumab, Brodalumab, Dupilumab, Durvalumab, Emicizumab, Guselkumab, lnotuzumab ozogamicin, Ocrelizumab, Sarilumab, Atezolizumab, Bezlotoxumab, Ixekizumab, Obiltoxaximab, Olaratumab, Reslizumab, Alirocumab, Daratumumab, Dinutuximab, Elotuzumab, Evolocumab, Mepolizumab, Necitumumab, Secukinumab, Nivolumab, Pembrolizumab, Ramucirumab, Siltuximab, Vedolizumab, Alemtuzumab, Obinutuzumab, Ado-trastuzumab emtansine, Pertuzumab, Raxibacumab, Belimumab, Brentuximab vedotin, Ipilimumab, Denosumab, Canakinumab, Golimumab, Ofatumumab, Tocilizumab, Ustekinumab, Eculizumab, Panitumumab, Bevacizumab, Cetuximab, Natalizumab, Omalizumab, Adalimumab, Ibritumomab tiuxetan, Basiliximab, Infliximab, Palivizumab, Trastuzumab, Rituximab, and Abciximab.

10. The cell according to claim 1, wherein the cell is modified to express a polypeptide product, wherein the polypeptide product comprises a component of a viral vector such as rAAV.

11. A method of modifying a cell, wherein the cell is modified to enhance β-1,4 galactosylation of a polypeptide product, the modifications comprising: reducing O-GalNAc galactosylation activity in the cell by reduction of functional COSMC molecular chaperone in the cell and/or by reduction of functional T-synthase in the cell; and modification of the cell to provide overexpression of β1,4-galactosyltransferase in the cell.

12. The method according to claim 11, wherein the method comprises the step of transforming the cell with nucleic acid encoding the β1,4-galactosyltransferase.

13. The method according to claim 11, wherein the method comprises modifying the gene(s) encoding the COSMC molecular chaperone and/or T-synthase, or modifying regulatory elements thereof in order to knockout the expression thereof.

14. The method according to claim 11, wherein the method further comprises modifying the cell to express a polypeptide product.

15. A method of producing a polypeptide product, the method comprising providing a cell according to claim 1, or obtaining a modified cell according to claim 11, and culturing the cell for expression of the polypeptide product.

16. The method according to claim 15, wherein the polypeptide product is produced to have at least 80% β-1,4 galactosylation and/or at least 50% bi-galactosylated; or wherein the polypeptide product is produced to have at least a 0.5 fold increased β-1,4 galactosylation and/or at least 0.5 fold increased bi-galactosylation.

17. The method according to any of claim 15 or 16, wherein the cells are cultured with a UMG feeding strategy comprising or consisting of the addition of uridine, manganese and galactose in the cell culture media.

18-20. (canceled)

21. A plasmid encoding: a genetic modification element(s) that is targeted to knock-out or reduce functional COSMC molecular chaperone and/or T-synthase expression in a cell; b4GalT for expression in the cell; and optionally a selection marker, such as an antibiotic resistance gene.

22. A kit comprising: a first plasmid encoding: a genetic modification element(s) that is targeted to knock-out or reduce functional COSMC molecular chaperone and/or T-synthase expression in a cell; and a second plasmid encoding b4GalT for expression in a cell and optionally a selection marker, such as an antibiotic resistance gene.

23. The kit according to claim 22, further comprising a plasmid encoding a polypeptide product for expression.

24. The kit according to claim 22, wherein the genetic modification element(s) comprise zinc finger nuclease, TALEN (transcription activator-like effector nuclease), guide RNA (gRNA) and Cas9 (for CRISPR modification), silencing RNAs, or homologous recombination cassettes with antibiotic resistance genes.

Description

[0103] FIG. 1—Sources of mAb N-glycan variability. Variability arises from the glycosylation process. Mechanisms: residence time in Golgi (q.sub.p).sup.1, enzyme activity, enzyme accessibility, etc. Metabolism: Nucleotide Sugar Donor (NSD) availability.sup.2. NSDs are simultaneously consumed for cellular & product glycosylation.

[0104] FIG. 2—N-glycan variability.

[0105] FIG. 3—Increase the mAb galactosylation capacity of CHO cells by: A. Eliminating galactosylation by knocking out the COSMC molecular chaperone; and B. Increasing cellular galactosylation capacity by overexpressing β1,4-galactosyltransferase I.

[0106] FIG. 4—CHO DP12 Cell Line. Both cell engineering events are required.

[0107] FIG. 5—CHO VRC01 Cell Line. Both cell engineering events are required.

[0108] FIG. 6—Comparison with other technologies.

[0109] FIG. 7—Fed-batch CHO DP12 cell culture. GalMAX enhances galactosylation

Example 1—GalMAX—Maximising Therapeutic Protein Galactosylation: Simultaneous Removal of Metabolic and Cellular Machinery Bottlenecks

SUMMARY

[0110] The positive impact of β-1,4 galactosylation on therapeutic protein efficacy has only been reported recently. Table 1 summarises the effects of various glycan modifications on mAb cancer killing ability, and highlights the positive impact galactosylation on safety, CDC, ADCC and PK/PD.

TABLE-US-00001 TABLE 1 mAb cancer killing ability. (1,4)-Galactosylation is the source of therapeutic protein variability. Galactosylation increases efficacy and reduces heterogeneity (see Raju et al (2012) mAbs 4(3): 385-391 Planinc et al. (2017) Eur. J. Hosp. Pharm. Sci. Pract. 24(5): 286-292, which is herein incorporated by reference). Glycan Safety CDC ADCC PK/PD Oligomannose — Negative Positive and Negative impact negative impact No impact Fucose — No impact Highly No impact negative impact Galactose Positive Positive Positive Positive impact impact impact impact Bisected — — Positive — impact NANA Positive — Negative Positive sialylated impact impact impact NGNA Highly negative — — Positive sialylated impact impact αGal epitope Highly negative — — Negative impact impact

[0111] The invention maximises the β-1,4 galactosylation of asparagine(N)-linked glycans of therapeutic monoclonal antibodies (mAbs) produced in cells (such as Chinese Hamster Ovary cells, CHO). The invention combines two genetic modifications to simultaneously eliminate metabolic bottlenecks (by increasing the intracellular availability of UDP-Gal, which is the co-substrate required for β-1,4 galactosylation) and cellular machinery bottlenecks (by increasing the amount of β4GalT1 expressed by the cells). The invention comprises the following two genetic engineering events, which can be performed sequentially or simultaneously:

[0112] 1. Genetic knockout of Core 1 β3GalT specific molecular chaperone (COSMC).

[0113] The first genetic engineering event involves eliminating the expression of COSMC, using a specific zinc finger nuclease [1] or CRISPR-Cas9 technology [2]. Abrogation of COSMC expression eliminates threonine/serine(O)-linked β-1,3 galactosylation, thus yielding cellular glycoproteins bearing the so-called Tn antigen. Eliminating O-linked galactosylation greatly reduces (by approximately 30% [3]) the consumption of uridine diphosphate galactose (UDP-Gal), the donor metabolite required for all galactosylation reactions, towards cellular galactosylation and increases UDP-Gal availability towards N-linked β-1,4 galactosylation of the therapeutic protein product.

[0114] 2. Genetic overexpression of the β-1,4 galactosyltransferase (β4GalT) enzyme.

[0115] The second genetic engineering event involves stably transfecting cells with the gene for β4GalT, which can be of any isotype (1 through 7) and sourced from different organisms (Homo sapiens, Mus musculus, Rattus novergicus, Pan troglodytes, Bos taurus, etc.). Preference is given to human β4GalT1 (UniProtKB protein P15291), as this particular enzyme has been reported to achieve the highest levels of β-1,4 galactosylation in CHO-derived glycoproteins [4].

[0116] Ectopic expression of the β4GalT enzyme increases the capacity and rate with which the cells add β-1,4-linked galactose residues to glycoprotein N-linked glycans.

[0117] Materials

[0118] The CHO DP-12 clone #1934 [aIL8.92 NB 28605/14] (ATCC® CRL-12445™) producing an anti-IL-8 IgG1κ was used for our preliminary studies. This cell line was adapted to grow in suspension using Ex-Cell® 302 serum-free media (Sigma-Aldrich, Cat. No. 14324C) with 4 mM of L-glutamine and 200 nM of MTX. Fed-batch culture was performed by supplementing the basal media (Sigma-Aldrich, Cat. No. 14324C) with 6.25% v/v of Ex-Cell Advanced CHO feed with glucose (Sigma-Aldrich, Cat. No. 24367C) every 48 hours, starting from day 3. The feed was further supplemented with 45% w/v glucose to maintain residual glucose concentration above 4 g/L after feeding. A second CHO cell line (VRC01), which is derived from CHO-K1 and produces a human IgG1κ, was used to confirm the GalMAX strategy. This cell line was adapted for suspension growth in serum-free ActiPro culture media (HyClone Cat. No. SH31039.02) supplemented with 4 mM L-glutamine and 100 nM MTX.

[0119] Ectopic expression of human β4GalT1 was performed by transfecting the DP-12 and VRC01 CHO cells with the pUNO1 plasmid bearing the coding sequence of human β4GalT1 (Invivogen Cat. Code puno1-hb4galt1) using an Amaxa® CLB-Transfection Device and an Amaxa® CLB-Transfection Kit (Lonza, Cat. No. VECA-1001), as per the manufacturer's instructions. The pUNO1 scaffold also contains the coding sequence for a blasticidin resistance gene, which was used to select successfully transfected cells.

[0120] Knocking out of COSMC was performed using the CRISPR-Cas9 genome editing system using an all-in-one pX458 plasmid (pSpCas9(BB)-2A-GFP) obtained from Addgene (plasmid #48138) [8]. The gRNA sequence (GAATATGTGAGTGTGGATGGAGG) targeting the C1GalT1C1 gene (Gene ID: 2375260) was designed using the CHO Cas9 Target Finder (http://staff.biosustain.dtu.dk/laeb/crispy, target ID 2375260) [6].

[0121] Cells successfully transfected with the pX458+sgRNA plasmid were selected for eGFP fluorescence using a FACSAria III Cell Sorter (Beckton-Dickinson) with a 488 nm (blue) excitation laser. Homogeneous populations were gated using forward and side scatter criteria to select singlets. Fluorescent-positive cells were identified using non-transfected cells as the auto-fluorescence control. Cell pools were recovered and cultured. After two weeks, the population of GFP-negative cells were isolated and cultured for an additional three days, after which, cells bearing the COSMC knockout phenotype (bearing only exposed GalNAc residues—the Tn antigen—on their surface O-linked glycans) were identified and isolated through lectin-aided cell sorting using the FACSAria III instrument.

[0122] The FITC-labelled Vicia villosa lectin (Vector Labs, Cat. No. B-1235), which is specific for the Tn antigen, was used for COSMC knockout cell sorting. A similar procedure was performed for cells transfected with human β4GalT1, but the lectin used was (fluorescein labelled lectin from Erythrina cristagalli, Vector Labs, Cat. No. FL-1141) to select cells presenting enhanced cell surface β-1,4-galactosylated glycans.

[0123] mAb glycan analysis was performed using a recently-developed method that has been optimised for quantification of glycopeptides (Carillo et al. Journal of Pharmaceutical Analysis. Volume 10, Issue 1, February 2020, Pages 23-34. https://doi.org/10.1016/j.jpha.2019.11.008, which is herein incorporated by reference).

[0124] Study Results

[0125] We have performed proof-of-concept studies in mAb-producing CHO cells where the invention yielded an increase in product β-1,4 galactosylation from 55.2% to 96.2% (CHO DP12) and from 45% to 97.6% (CHO VRC01), including a substantial increase in bi-galactosylated species from 8.4% to 79.4% (CHO DP12) and 5.2% to 80.3% (CHO VRC01) (Table 8 and FIGS. 4 and 5). A key finding confirming the rationale underlying the designed technology is that the simultaneous knock out of COSMC and ectopic expression of human βGalT1 are required to achieve the exceptional increases in product β-1,4 galactosylation obtained with the invention.

[0126] To confirm the advantages of the technology, CHO-DP12 was cultured under fed-batch operation where the invention yielded an increase in product β-1,4 galactosylation from 47.7% to 91.8%, including a substantial increase in bi-galactosylated species from 7.2% to 80.0% (Table 9 and FIG. 7). The two key findings are that (i) the invention produces enhanced product β-1,4 galactosylation under fed-batch culture, which is standard practice for large scale mAb manufacturing and (ii) that the invention yields substantially higher galactosylation than the cells overexpressing only β4GalT1, thus indicating that both genetic modifications are required to maximise product β-1,4 galactosylation.

[0127] This invention provides a simple and robust solution for key Quality Assurance challenges that arise during the manufacture of recombinant therapeutic glycoproteins, including monoclonal antibodies (mAbs). The key challenges tackled by the invention are those associated (1) the heterogeneity, (2) the therapeutic efficacy (pharmacodynamics) and (3) the serum half-life (pharmacokinetics) of therapeutic glycoproteins, as outlined below.

[0128] 1. Maximising β-1,4 Galactosylation Reduces the Heterogeneity of Therapeutic Proteins.

[0129] β-1,4 galactosylation is the major source of therapeutic protein variability [10, 11] and arises from even subtle process deviations during manufacturing operations [12]. Nutrient availability, metabolite accumulation, temperature, pH and other bioprocess conditions vary during the large-scale cell culture processes whereby therapeutic proteins are manufactured. Variations in bioprocess conditions are widely known to heavily influence glycosylation, in particular, β-1,4 galactosylation [13]. Reducing product heterogeneity is key to ensuring the safety and therapeutic efficacy of therapeutic proteins and is, thus, a fundamental aim of biopharmaceutical manufacturing operations.

[0130] The invention neutralises the negative effects of varying bioprocess conditions on product β-1,4 galactosylation, thus reducing product heterogeneity: [0131] The nutrient availability issues are addressed by eliminating the consumption of UDP-Gal towards mAb-irrelevant cellular O-linked galactosylation, which accounts for approximately 30% of all UDP-Gal consumed in non-engineered CHO cells [7]. When cellular O-galactosylation is eliminated, more UDP-Gal is made available and product N-galactosylation becomes less dependent on fluctuations in nutrient availability. [0132] The invention reduces the negative impact of metabolite accumulation (e.g. ammonia) or pH shifts/excursions, both of which reduce β4GalT activity, through genetic overexpression of the enzyme. This reduces the sensitivity to changes in the cell culture environment and, in consequence, also reduces product heterogeneity.

[0133] 2. Increased β-1,4 Galactosylation Enhances the Efficacy of Both Anti-Cancer and Anti-Inflammatory Monoclonal Antibodies (mAbs).

[0134] High levels of β-1,4 galactosylated mAb glycans increases antibody-dependent cellular cytotoxicity (ADCC) by up to 30% [14] and complement-dependent cellular cytotoxicity (CDC) by up to 26% [15]. In many commercial products, both cytotoxic mechanisms (ADCC and CDC) define the therapeutic efficacy of anti-cancer mAbs [16, 17].

[0135] The invention achieves unprecedented levels of β-1,4 galactosylation on the Fc of mAbs produced by CHO cells—up to 98% β4-galactosylated N-glycans (Table 8 and FIG. 6), thus demonstrating great potential in enhancing the anti-cancer activity of mAb products.

[0136] The presence of α-2,6-N-acetylneuraminic acid (α6Neu5Ac) residues on the glycans of antibody therapies have been reported to enhance immune modulation and, thus, increase the efficacy of anti-inflammatory mAbs. Because the addition of α6Neu5Ac requires β-1,4 galactose residues as acceptors, high levels of β-1,4 galactosylation enable increased presence of α6Neu5Ac [19-21]; therefore, the increased levels of product β-1,4 galactosylation achieved by the invention may positively contribute to enhanced efficacy of anti-inflammatory mAbs.

[0137] 3. Increased β-1,4 Galactosylation Contributes to Enhancing the Serum Half-Life of Therapeutic Proteins.

[0138] Therapeutic glycoproteins with higher Neu5Ac content (sialylation) require reduced doses or less frequent dosing because increased sialylation extends the half-life of therapeutic glycoproteins in the patient's serum [22, 23]. Increased sialylation may therefore reduce treatment costs for healthcare providers and patients. Again, β-1,4 galactosylation is intrinsically necessary for the addition of Neu5Ac residues. Therefore, increased levels of β-1,4 galactosylation are required to achieve enhanced serum half-life and, thereby, to improved therapeutic efficacy of therapeutic glycoproteins.

[0139] CHO DP12 Cell Line

[0140] Referring to FIG. 4 and Tables 2 and 3—CHO DP12 Cell Line. Both cell engineering events are required.

[0141] FIG. 4 and Tables 2 and 3 present the distribution of glycans present on the Fc of mAbs produced with six cell lines derived from parental CHO DP12 cells: [0142] (i) CHO DP12 Parental: non-engineered CHO DP12 cells. [0143] (ii) CHO DP12 mCOSMC−: CHO DP12 cells transfected with the CRISPR plasmid in absence of the gRNA sequence targeting COSMC (Mock COSMC− plasmid). [0144] (iii) CHO DP12 COSMC−: CHO DP12 cells transfected with the CRISPR plasmid containing the COSMC-targeting gRNA. [0145] (iv) CHO DP12 mGalT+: CHO DP12 cells transfected with the empty (absence of hβ4GalT1) pUNO plasmid (Mock GalT+ plasmid). [0146] (v) CHO DP12 GalT+: CHO DP12 cells transfected with the hβ4GalT1-containing pUNO plasmid. [0147] (vi) CHO DP12 GalMAX (COSMC−/GalT+): CHO DP12 cells that simultaneously contain the COSMC knockout and hβ4GalT1 expression (transfected/selected for CRISPR knockout of COSMC and transfected/selected for hβ4GalT1 expression).

[0148] FIG. 4A presents the mAb Fc glycoform distributions produced by each of the six CHO DP12 cell lines cultured under batch conditions. The data correspond to triplicate cultures for CHO DP12 Parental, duplicate cultures for mCOSMC−, four cultures for COSMC−, five cultures for mGalT+, duplicate cultures for GalT+ and four cultures for CHO DP12 GalMAX. The mAb Fc glycosylation patterns were measured using mass spectrometry (Carillo et al. Journal of Pharmaceutical Analysis. Volume 10, Issue 1, February 2020, Pages 23-34. https://doi.org/10.1016/j.jpha.2019.11.008).

[0149] Four major glycoforms are observed: [0150] (i) A2G0F: biantennary, non galactosylated and fucosylated. [0151] (ii) A2G1F: biantennary, mono-galactosylated and fucosylated. [0152] (iii) A2G2F: biantennary, bi-galactosylated and fucosylated. [0153] (iv) A2S1G2F: biantennary, mono-sialylated, bi-galactosylated and fucosylated.

[0154] There are no statistically significant differences (two-tailed t-test) among the mAb Fc glycoprofiles produced by the CHO DP12 Parental, CHO DP12 mCOSMC−, CHO DP12 COSMC−, and CHO DP12 mGalT+ cell lines, where the average relative abundances of A2G0F, A2G1F and A2G2F are 47.8%±5%, 43.8%±4.1%, and 8.3%±1.4%, respectively (averages and standard deviations across all replicate cultures for the four cell lines).

[0155] The cell lines expressing hβ4GalT1 present a substantial decrease in A2G0F and a concomitant increase in A2G2F. For the CHO DP12 GalT+ cell line, A2G0F is reduced to 7.5%±0.3% and A2G2F increases to 77.9%±1.5%. For the CHO DP12 GalMAX (COSMC−/GalT+) cell line, A2G0F is reduced to 3.8%±0.4% and A2G2F increases to 79.4%±1.8%. Although no statistically significant difference is observed in the bi-galactosylated A2G2F glycoform produced by the GalT+ and GalMAX cell lines, CHO DP12 GalMAX cells produce approximately half of the non-galactosylated A2G0F glycoform generated by GalT+ cells (p<0.01), thus indicating that the knockout of COSMC contributes to increased mAb galactosylation and overall glycan homogeneity.

[0156] FIG. 4B presents a comparison between the total non-galactosylated (A2G0F) and galactosylated (sum of A2G1F, A2G2F and A2S1G2F) glycoforms produced by all CHO DP12 cell lines. Again, there is no statistically significant difference in mAb Fc galactosylation among the Parental, COSMC− (Mock), COSMC−, and GalT+(Mock) cell lines, where overall averages (across all four cell lines) for non-galactosylation and galactosylation are 47.8%±5.0% and 52.2%±5.0%, respectively. In contrast, a significant difference in galactosylation is observed between the CHO DP12 GalT+ and GalMAX cell lines, where CHO DP12 GalMAX produces 3.8%±0.4% non-galactosylated and 96.2%±0.6% galactosylated species compared to 7.5%±0.3% and 92.5%±0.3% produced by the CHO DP12 GalT+ cell line. Overall, when deployed in the CHO DP12 cell line, the GalMAX invention delivers a 12-fold decrease in A2G0F and a 9.4-fold increase in A2G2F as well as a 74.1% increase in galactosylated glycoforms, when compared to the parental cell line.

[0157] Table 2 provides numerical values for FIG. 4A.

TABLE-US-00002 Relative abundance ± S.D. (%) A2G0F A2G1F A2G2F DP12 Parental 44.7 ± 3.1 46.8 ± 2.6 8.4 ± 0.8 DP12 mCOSMC− 49.6 ± 4.2 43.0 ± 2.2 7.1 ± 1.7 DP12 COSMC− 45.0 ± 6.3 45.8 ± 5.5 9.1 ± 0.9 DP12 mGalT+ 51.2 ± 2.4 40.6 ± 1.3 8.1 ± 1.5 Average 47.8 ± 5.0 43.8 ± 4.1 8.3 ± 1.4 DP12 GalT+  7.5 ± 0.3 12.8 ± 0.2 77.9 ± 1.5  DP12  3.8 ± 0.4 15.5 ± 1.9 79.4 ± 1.8  GalMAX 12-fold reduction 9.4-fold increase

[0158] Table 3 provides numerical values for FIG. 4B.

TABLE-US-00003 Relative abundance ± S.D. (%) Non-galactosylated Galactosylated DP12 Parental 44.7 ± 3.1 55.2 ± 3.2 DP12 mCOSMC− 49.6 ± 4.2 50.2 ± 4.1 DP12 COSMC− .sup. 45 ± 6.3 55.2 ± 6.3 DP12 mGalT+ 51.2 ± 2.4 48.8 ± 2.4 Average 47.8 ± 5.0 52.2 ± 5.0 DP12 GalT+  7.5 ± 0.3 92.5 ± 0.3 DP12  3.8 ± 0.4 96.2 ± 0.6 GalMAX 12-fold reduction 74.1% increase

[0159] CHO VRC01 Cell Line

[0160] Referring to FIG. 5 and Tables 4 and 5—CHO VRC01 Cell Line. Both cell engineering events are required.

[0161] FIG. 5 and Tables 4 and 5 present the distribution of glycans present on the Fc of mAbs produced with six CHO VRC01-derived cell lines, which, similarly to their DP12 counterparts, are named VRC01 Parental, VRC01 mCOSMC−, VRC01 COSMC−, VRC01 mGalT+, VRC01 GalT+ and VRC01 GalMAX (COSMC−/GalT+).

[0162] FIG. 5A presents the mAb Fc glycoform distributions produced by each of the six CHO VRC01 cell lines cultured under batch conditions. The data correspond to five cultures of VRC01 Parental, a single culture of VRC01 mCOSMC−, four cultures of VRC01 COSMC−, duplicate cultures of VRC01 mGalT+, five cultures of VRC01 GalT+ and triplicate cultures of VRC01 GalMAX. The mAb Fc glycosylation patterns were measured using mass spectrometry method outlined by Carillo et al. (Journal of Pharmaceutical Analysis. Volume 10, Issue 1, February 2020, Pages 23-34. https://doi.org/10.1016/j.jpha.2019.11.008).

[0163] The same major glycoforms produced by CHO DP12-derived cell lines are also observed for the CHO VRC01 cell lines (A2G0F, A2G1F, A2G2F, and A2S1G2F). Little or no statistically significant differences (two-tailed t-test) were observed among the mAb Fc glycoprofiles produced by the VRC01 Parental, VRC01 mCOSMC−, VRC01 COSMC−, and VRC01 mGalT+ cell lines, where the average relative abundances of A2G0F, A2G1F and A2G2F are 58.0%±37.4%±4.7%, and 4.6%±1.1%, respectively (averages and standard deviations across all replicate cultures for the four cell lines).

[0164] The cell lines expressing hβ4GalT1 present a substantial decrease in A2G0F and a concomitant increase in A2G2F. For the VRC01 GalT+ cell line, A2G0F is reduced to 8.6%±5.0% and A2G2F increases to 72.0%±5.8%. In the VRC01 GalMAX cell line, A2G0F is reduced to 2.4%±0.4% and A2G2F increases to 80.3%±0.6%. The VRC01 GalMAX cell line produces 3.6±2.2-fold less A2G0F than the VRC01 GalT+ cell line, thus indicating that the knockout of COSMC contributes to increased mAb galactosylation and overall glycan homogeneity.

[0165] FIG. 5B presents a comparison between the total non-galactosylated (A2G0F) and galactosylated (sum of A2G1F, A2G2F and A2S1G2F) glycoforms produced by all CHO VRC01 cell lines. There are small differences among the mAb Fc glycoprofiles generated by the Parental, mCOSMC−, COSMC−, and mGalT+ cell lines, where overall averages (across all four cell lines) for non-galactosylation and galactosylation are 58.0%±5.7% and 42.0±5.5%, respectively. The cell lines expressing hβ4GalT1 present substantial increases in galactosylation. VRC01 GalT+produces 91.4%±5.0% galactosylated and 8.6%±5.0% non-galactosylated mAb Fc glycans. VRC01 GalMAX generates 97.6%±0.4% galactosylated and only 2.4%±0.4% non-galactosylated mAb Fc glycans. Overall, the GalMAX invention deployed in VRC01 cells delivers a 2.2-fold increase in galactosylation and a 23-fold decrease in non-galactosylated glycans when compared to the parental VRC01 cell line.

[0166] Table 4 provides numerical values for FIG. 5A.

TABLE-US-00004 Relative abundance ± S.D. (%) A2G0F A2G1F A2G2F VRC01 Parental 55.0 ± 2.6 39.8 ± 2.1 5.2 ± 0.7 VRC01 mCOSMC− 59.9 35.8 4.3 VRC01 COSMC− 58.3 ± 8.3 37.2 ± 5.7 4.5 ± 1.1 VRC01 mGalT+ 63.9 ± 1.9 32.6 ± 1.6 3.5 ± 0.3 Average 58.0 ± 5.7 37.4 ± 4.7 4.6 ± 1.1 VRC01 GalT+  8.6 ± 5.0 18.5 ± 1.6 72.0 ± 5.8  VRC01 GalMAX  2.4 ± 0.4 16.3 ± 1.1 80.3 ± 0.6  23-fold reduction 15.5-fold increase

[0167] Table 5 provides numerical values for FIG. 5B.

TABLE-US-00005 Relative abundance ± S.D. (%) Non-galactosylated Galactosylated VRC01 Parental 55.0 ± 2.6 45.0 ± 2.6 VRC01 mCOSMC− 59.9 40.1 VRC01 COSMC− 58.3 ± 8.3 41.7 ± 8.3 VRC01 mGalT+ 63.9 ± 1.9 36.1 ± 1.9 Average 58.0 ± 5.7 42.0 ± 5.5 VRC01 GalT+  8.6 ± 5.0 91.4 ± 5.0 VRC01 GalMAX  2.4 ± 0.4 97.6 ± 0.4 23-fold reduction 2.2-fold increase

[0168] The results obtained for the CHO DP-12 and VRC01 cell lines are consistent with the mechanisms governing galactosylation. The two key bottlenecks which limit galactosylation are (i) UDP-Gal availability and (ii) β4GalT availability/activity. If there is sufficient UDP-Gal, but β4GalT availability/activity is reduced, then low levels of galactosylation will be observed. Conversely, if β4GalT availability/activity is in excess but there is insufficient UDP-Gal availability, low levels of galactosylation will also be observed. Our overall results indicate that β4GalT availability limits galactosylation in the cell lines where hβ4GalT1 is not expressed (Parental, mCOSMC−, COSMC−, and mGalT+). This bottleneck is alleviated when hβ4GalT1 is ectopically expressed (GalT+ and the GalMAX cell lines), and the increase in overall galactosylation between the GalT+ and GalMAX cell lines is due to the gain in UDP-Gal availability afforded by the COSMC knockout. Therefore, both cell engineering interventions (COSMC knockout and ectopic h(34GalT1 expression) are required to maximise mAb Fc galactosylation and, thus, the GalMAX invention is validated.

[0169] Table 6 provides a comparison of Integral of Viable Cells (IVC), specific productivity (q.sub.p) and product titre for batch cultures of CHO DP12 and CHO VRC01.

TABLE-US-00006 IVC q.sub.p (10.sup.6 cells (pg cell.sup.−1 Titre mL.sup.−1 day) day.sup.−1) (mg L.sup.−1) DP12 Parental 14.3 ± 0.2 6.2 ± 0.2 123.1 ± 0.1 DP12 COSMC− 16.1 ± 0.1 5.9 ± 0.0 128.9 ± 1.7 DP12 GalT+ 16.5 ± 0.3 4.3 ± 0.0 104.8 ± 1.2 DP12 GalMAX 19.5 ± 1.0 4.0 ± 0.0 133.5 ± 0.2 VRC01 Parental 48.3 ± 0.9 5.0 ± 0.3 297.4 ± 3.8 VRC01 COSMC− 46.5 ± 0.8 5.8 ± 0.6 314.4 ± 5.2 VRC01 GalT+ 49.5 ± 0.1 5.6 ± 0.4 327.5 ± 2.0 VRC01 GalMAX 47.4 ± 1.1 6.4 ± 0.7 333.6 ± 5.3

[0170] Table 7 provides a comparison of Integral of Viable Cells (IVC), specific productivity (q.sub.p) and product titre for fed-batch cultures of CHO DP12.

TABLE-US-00007 IVC q.sub.p Titre (10.sup.6 cells mL.sup.−1 d) (pg cell.sup.−1 day.sup.−1) (mg L.sup.−1) DP12 Parental 43.6 ± 1.2 2.1 ± 0.0 155.6 ± 1.5 DP12 COSMC− 43.0 ± 0.4 2.7 ± 0.0 182.6 ± 0.6 DP12 GalT+ 37.4 ± 0.4 1.4 ± 0.1 109.0 ± 5.0 DP12 GalMAX 37.7 ± 0.1 2.5 ± 0.1 157.6 ± 3.8

[0171] Tables 6 and 7 present a comparison of cell culture KPIs to demonstrate that the GalMAX technology has no negative impact on the productivity of the CHO DP12 and CHO VRC01 cell lines used for the study. Table 6 presents data corresponding to batch cultivation, where DP12 GalMAX presents an increase in integral of viable cells (IVC) from 14.3±0.22 to 19.5±0.95 10.sup.6 cells mL.sup.−1 day when compared with the parental cell line. This increase in IVC results in a reduction in specific productivity (q.sub.p) from 6.21±0.16 to 4.04±0.02 pg cell.sup.−1 day.sup.−1, when compared with parental CHO DP12. These changes in IVC and q.sub.p offset to yield similar titres. DP12 GalMAX achieves a mAb titre of 133.5±0.2 mg L.sup.−1, which is slightly higher than the 123.1±0.1 mg L.sup.−1 produced by Parental CHO DP12.

[0172] The VRC01 GalMAX cell line yield no statistically significant difference in values for IVC when compared with the VRC01 Parental (47.4±1.1 10.sup.6 cells mL.sup.−1 day and 48.3±0.9 10.sup.6 cells mL.sup.−1 day, respectively). Interestingly, VRC01 GalMAX presents an increased q.sub.p (6.4±0.7 pg cell.sup.−1 day.sup.−1), when compared with parental CHO DP12 (5.0±0.3 pg cell.sup.−1 day.sup.−1). The increase in q.sub.p alongside the similar IVC yields a slight increase in product titre achieved by the VRC01 GalMAX cell line, when compared to the VRC01 parental (333.6±5.3 mg L.sup.−1 vs. 297.4±3.8 mg L.sup.−1, respectively).

[0173] Table 7 compares IVC, q.sub.p and titre across four CHO DP12-derived cell lines (DP12 Parental, DP12 COSMC−, DP12 GalT+, and DP12 GalMAX) when cultured under fed-batch mode. DP12 GalMAX achieves a slightly lower IVC of 37.7±0.1 10.sup.6 cells mL.sup.−1 day when compared to the 43.6±1.2 10.sup.6 cells mL.sup.−1 day achieved by DP12 Parental. This decreased IVC results in a slightly increased q.sub.p of 2.5±0.1 pg cell.sup.−1 day.sup.−1 when compared to the 2.1±0.0 pg cell.sup.−1 day.sup.−1 of the DP12 parental cell line. These differences in IVC and q.sub.p offset to yield similar titres of 157.6±3.8 mg L.sup.−1 (DP12 GalMAX) and 155.6±1.5 mg L.sup.−1 (DP12 Parental).

[0174] Overall, Tables 6 and 7 demonstrate that the GalMAX technology has no negative impact on cell culture KPIs—in all cases, the GalMAX technology yielded titres that are higher than the parental cell lines.

[0175] Comparison of GalMAX with Other Technologies

[0176] Referring to FIG. 6 and Table 8. GalMAX performs comparably or outperforms other available technologies.

[0177] FIG. 6 and Table 8 compare the GalMAX invention with other technologies that have been developed to maximise mAb Fc galactosylation. UMG feeding refers to uridine-manganese-galactose cell culture supplementation [1, 2], β4GalT expression refers to ectopic expression of the β4GalT enzyme [19, 24, 25], and Enzymatic remodelling refers to in vitro enzymatic modification of mAb Fc glycans [4, 14].

[0178] The GalMAX invention delivers between 96.2% and 97.6% overall galactosylation (DP12 and VRC01 cell lines, respectively). These values are comparable with the 98% total galactosylation achieved by the cell engineering strategy of Schulz et al. [25] and the in vitro methods of Thomann et al. [14] and Tayi & Butler [4]. GalMAX considerably outperforms the cell engineering strategies by Raymond et al. and Chang et al. [24], which yield values of 73% and 87%, respectively. GalMAX also outperforms UMG feeding strategies, which report between 48% and 67% total mAb Fc galactosylation [1, 2].

[0179] The GalMAX invention yields up to 80% bi-galactosylated mAb Fc glycans, which is comparable with the value of 83% obtained by Thomann et al. [14] (in vitro). The levels of mAb Fc bi-galactosylation obtained by Schulz et al. [25] (cell engineering) and Tayi & Butler (in vitro) are slightly higher than those achieved by the GalMAX invention. GalMAX produces a considerably larger fraction of bi-galactosylated mAb Fc glycans than those reported for UMG feeding [1, 2], and other cell engineering strategies [19, 24].

[0180] Table 8 compares the performance of GalMAX with existing technologies.

TABLE-US-00008 Total galactosylation Total bi-galactosylation After Fold After Fold Titre Initial Treatment Increase Initial Treatment Increase (g/L) Refs. UMG feeding 9.4% 48.1% 5.1 0.5% 9.7% 19.4 3.20  [1] 35.6% 67.1% 1.9 4.5% 19.1% 4.2 0.32  [2] β4GalT 37.4% 72.9% 2.0 5.1% 48.8% 9.6 0.02 [19] expression 10.2% 86.7% 8.5 1.4% 62.3% 44.5 0.05 [24] 45.1% 97.9% 2.2 6.8% 87.2% 12.8 0.30 [25] Enzymatic 40.3% 98.2% 2.4 4.4% 82.8% 18.8 N/A [14] remodelling 39.0% 98.0% 2.5 5.0% 92.5% 18.5 N/A  [4] DP12 GalMAX 55.2% 96.2% 1.7 8.4% 79.4% 9.4 0.13 VRC01 GalMAX 45.0% 97.6% 2.2 5.2% 80.3% 15.5 0.33

[0181] Fed-Batch Culture of CHO DP12

[0182] Referring to FIG. 7 and Table 9. GalMAX CHO DP12 Cell Line. Both cell engineering events are required and the level of mono- and bi-galactosylation is maintained across batch and fed-batch culture.

[0183] FIG. 7 and Table 9 present the distribution of glycans present on the Fc of mAbs produced with the four engineered cell lines derived from CHO DP12 (DP12 Parental, DP12 COSMC−, DP12 GalT+ and DP12 GalMAX) cultured under fed-batch mode. The data correspond to duplicate cultures of each cell line. The mAb Fc glycosylation patterns were measured using mass spectrometry method outlined by Carillo et al. (Journal of Pharmaceutical Analysis. Volume 10, Issue 1, February 2020, Pages 23-34. https//doi.org/10.1016/j.jpha.2019.11.008).

[0184] FIG. 7A shows that the same major glycoforms observed for batch culture are also observed for fed-batch mode (A2G0F, A2G1F, A2G2F, and A2S1G2F). The DP12 COSMC− cell line produces marginally lower non-galactosylated glycoform A2G0F and higher mono-galactosylated A2G1F glycans. No statistically significant differences are observed in the fraction of bi-galactosylated A2G2F glycan. These results indicate that COSMC knockout contribute to enhanced product glycosylation in cells cultured under fed-batch operation.

[0185] FIG. 7A shows that, as with batch cultivation, CHO DP12 expressing hβ4GalT1 present a substantial decrease in A2G0F (from 51.4±1.2 to 11.8±2.0) and a concomitant increase in A2G2F (from 7.2±0.3 to 57.5±8.6). In the CHO DP12 GalMAX cell line, A2G0F is reduced to 1.7%±0.2% and A2G2F increases to 80.0%±0.6%. The DP12 GalMAX cell line produces 7.1±1.5-fold less A2G0F than the DP12 GalT+ cell line, thus indicating that the knockout of COSMC substantially contributes to increased mAb galactosylation and overall glycan homogeneity.

[0186] FIG. 7B presents a comparison between the total non galactosylated (Man5+A2G0F) and galactosylated (sum of A2G1F, A2G2F and A2S1G2F) glycoforms produced by the four DP12-derived cell lines when cultured in fed-batch mode. Minor differences are observed between the galactosylation of product generated by DP12 Parental and DP12 COSMC− cells (47.7%±1.2% and 50.7%±1.4%, respectively). DP12 GalT+produces 77.1%±11.0% galactosylated and 22.6%±8.1% non-galactosylated mAb Fc glycans. DP12 GalMAX generates 91.8%±0.3% galactosylated and 8.2%±0.2% mAb Fc glycans. Overall, the GalMAX invention deployed in CHO DP12 cells cultured under fed-batch mode yield a 1.9-fold increase in galactosylation and a 6.4-fold decrease in non-galactosylated glycans when compared to the parental DP12 cell line.

[0187] Table 9 provides numerical values for FIG. 7A.

TABLE-US-00009 Relative abundance ± S.D. (%) A2G0F A2G1F A2G2F DP12 Parental 51.4 ± 1.2 40.5 ± 0.9  7.2 ± 0.3 DP12 COSMC− 48.0 ± 0.9 43.2 ± 0.6  7.5 ± 0.8 DP12 GalT+ 11.8 ± 2.0 18.9 ± 1.4 57.5 ± 8.6 DP12 GalMAX  1.7 ± 0.2 11.1 ± 0.2 80.0 ± 0.6 12-fold 3.6-fold 11-fold reduction reduction increase

DISCUSSION

[0188] The key aspect that sets the proposed invention aside from other cell glycoengineering strategies is the combination of knocking out COSMC expression and ectopically expressing β4GalT1.

[0189] Knocking out COSMC provides additional UDP-Gal co-substrate required for product β-1,4 galactosylation without additional manipulations or feeding strategies that are known to negatively impact cell growth and productivity. Computational studies from our group indicate that cellular O-linked galactosylation is the largest sink for UDP-Gal consumption in CHO cells—over 30% of all UDP-Gal is consumed goes towards cellular O-linked galactosylation [7].

[0190] Simultaneously overexpressing the β4GalT1 enzyme provides additional cellular machinery to catalyse the reaction whereby β-1,4 galactose is added to product N-linked glycans. The baseline levels of endogenous β4GalTs in CHO cells are insufficient to perform extensive product β-1,4 galactosylation. Alongside this, commonly observed variations in cell culture conditions (e g ammonia accumulation or pH shifts) may negatively impact the activity of endogenous β4GalTs. Therefore, ectopically expressing β4GalT1 provides additional machinery to achieve higher product β-1,4 galactosylation and simultaneously limits the negative impacts varying cell culture conditions have on enzymatic activity.

[0191] β-1,4 galactosylation has only been recently identified as a key determining factor of the therapeutic efficacy of mAb products. Thus, there are great opportunities to enhance the pharmacokinetics and pharmacodynamics of these products by maximising this glycan motif. This invention presents an entirely novel and facile genetic engineering strategy that maximises the β-1,4 galactosylation of mAbs. In the context of biopharmaceutical manufacturing, the invention has broad scope of implementation and could rapidly contribute to enhancing the safety and efficacy of life-saving medicines that are the highest-grossing class of pharmaceutical products.

TABLE-US-00010 Sequences β1,4-galactosyltransferase I isoform 1 amino acid sequence >NP_001488.2 beta-1,4-galactosyltransferase 1 isoform 1 [Homo sapiens] (SEQ ID NO: 1) MRLREPLLSGSAAMPGASLQRACRLLVAVCALHLGVTLVY YLAGRDLSRLPQLVGVSTPLQGGSNSAAAIGQSSGELRTG GARPPPPLGASSQPRPGGDSSPVVDSGPGPASNLTSVPVP HTTALSLPACPEESPLLVGPMLIEFNMPVDLELVAKQNPN VKMGGRYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLHP VLQRQQLDYGIYVINQAGDTIFNRAKLLNVGFQEALKDYD YTCFVFSDVDLIPMNDHNAYRCFSQPRHISVAMDKFGFSL PYVQYFGGVSALSKQQFLTINGFPNNYWGWGGEDDDIFNR LVFRGMSISRPNAVVGRCRMIRHSRDKKNEPNPQRFDRIA HTKETMLSDGLNSLTYQVLDVQRYPLYTQITVDIGTPS β1,4-galactosyltransferase I DNA/encoding sequence >NM_001497.4 Homo sapiens beta-1,4-galactosyltransferase 1 (B4GALT1), transcript variant 1, mRNA (SEQ ID NO: 2) GCTCCCAGGTCTGGCTGGCTGGAGGAGTCTCAGCTCTCAG CCGCTCGCCCGCCCCCGCTCCGGGCCCTCCCCTAGTCGCC GCTGTGGGGCAGCGCCTGGCGGGCGGCCCGCGGGCGGGTC GCCTCCCCTCCTGTAGCCCACACCCTTCTTAAAGCGGCGG CGGGAAGATGAGGCTTCGGGAGCCGCTCCTGAGCGGCAGC GCCGCGATGCCAGGCGCGTCCCTACAGCGGGCCTGCCGCC TGCTCGTGGCCGTCTGCGCTCTGCACCTTGGCGTCACCCT CGTTTACTACCTGGCTGGCCGCGACCTGAGCCGCCTGCCC CAACTGGTCGGAGTCTCCACACCGCTGCAGGGCGGCTCGA ACAGTGCCGCCGCCATCGGGCAGTCCTCCGGGGAGCTCCG GACCGGAGGGGCCCGGCCGCCGCCTCCTCTAGGCGCCTCC TCCCAGCCGCGCCCGGGTGGCGACTCCAGCCCAGTCGTGG ATTCTGGCCCTGGCCCCGCTAGCAACTTGACCTCGGTCCC AGTGCCCCACACCACCGCACTGTCGCTGCCCGCCTGCCCT GAGGAGTCCCCGCTGCTTGTGGGCCCCATGCTGATTGAGT TTAACATGCCTGTGGACCTGGAGCTCGTGGCAAAGCAGAA CCCAAATGTGAAGATGGGCGGCCGCTATGCCCCCAGGGAC TGCGTCTCTCCTCACAAGGTGGCCATCATCATTCCATTCC GCAACCGGCAGGAGCACCTCAAGTACTGGCTATATTATTT GCACCCAGTCCTGCAGCGCCAGCAGCTGGACTATGGCATC TATGTTATCAACCAGGCGGGAGACACTATATTCAATCGTG CTAAGCTCCTCAATGTTGGCTTTCAAGAAGCCTTGAAGGA CTATGACTACACCTGCTTTGTGTTTAGTGACGTGGACCTC ATTCCAATGAATGACCATAATGCGTACAGGTGTTTTTCAC AGCCACGGCACATTTCCGTTGCAATGGATAAGTTTGGATT CAGCCTACCTTATGTTCAGTATTTTGGAGGTGTCTCTGCT CTAAGTAAACAACAGTTTCTAACCATCAATGGATTTCCTA ATAATTATTGGGGCTGGGGAGGAGAAGATGATGACATTTT TAACAGATTAGTTTTTAGAGGCATGTCTATATCTCGCCCA AATGCTGTGGTCGGGAGGTGTCGCATGATCCGCCACTCAA GAGACAAGAAAAATGAACCCAATCCTCAGAGGTTTGACCG AATTGCACACACAAAGGAGACAATGCTCTCTGATGGTTTG AACTCACTCACCTACCAGGTGCTGGATGTACAGAGATACC CATTGTATACCCAAATCACAGTGGACATCGGGACACCGAG CTAGCGTTTTGGTACACGGATAAGAGACCTGAAATTAGCC AGGGACCTCTGCTGTGTGTCTCTGCCAATCTGCTGGGCTG GTCCCTCTCATTTTTACCAGTCTGAGTGACAGGTCCCCTT CGCTCATCATTCAGATGGCTTTCCAGATGACCAGGACGAG TGGGATATTTTGCCCCCAACTTGGCTCGGCATGTGAATTC TTAGCTCTGCAAGGTGTTTATGCCTTTGCGGGTTTCTTGA TGTGTTCGCAGTGTCACCCCAGAGTCAGAACTGTACACAT CCCAAAATTTGGTGGCCGTGGAACACATTCCCGGTGATAG AATTGCTAAATTGTCGTGAAATAGGTTAGAATTTTTCTTT AAATTATGGTTTTCTTATTCGTGAAAATTCGGAGAGTGCT GCTAAAATTGGATTGGTGTGATCTTTTTGGTAGTTGTAAT TTAACAGAAAAACACAAAATTTCAACCATTCTTAATGTTA CGTCCTCCCCCCACCCCCTTCTTTCAGTGGTATGCAACCA CTGCAATCACTGTGCATATGTCTTTTCTTAGCAAAAGGAT TTTAAAACTTGAGCCCTGGACCTTTTGTCCTATGTGTGTG GATTCCAGGGCAACTCTAGCATCAGAGCAAAAGCCTTGGG TTTCTCGCATTCAGTGGCCTATCTCCAGATTGTCTGATTT CTGAATGTAAAGTTGTTGTGTTTTTTTTTAAATAGTAGTT TGTAGTATTTTAAAGAAAGAACAGATCGAGTTCTAATTAT GATCTAGCTTGATTTTGTGTTGATCCAAATTTGCATAGCT GTTTAATGTTAAGTCATGACAATTTATTTTTCTTGGCATG CTATGTAAACTTGAATTTCCTATGTATTTTTATTGTGGTG TTTTAAATATGGGGAGGGGTATTGAGCATTTTTTAGGGAG AAAAATAAATATATGCTGTAGTGGCCACAAATAGGCCTAT GATTTAGCTGGCAGGCCAGGTTTTCTCAAGAGCAAAATCA CCCTCTGGCCCCTTGGCAGGTAAGGCCTCCCGGTCAGCAT TATCCTGCCAGACCTCGGGGAGGATACCTGGGAGACAGAA GCCTCTGCACCTACTGTGCAGAACTCTCCACTTCCCCAAC CCTCCCCAGGTGGGCAGGGCGGAGGGAGCCTCAGCCTCCT TAGACTGACCCCTCAGGCCCCTAGGCTGGGGGGTTGTAAA TAACAGCAGTCAGGTTGTTTACCAGCCCTTTGCACCTCCC CAGGCAGAGGGAGCCTCTGTTCTGGTGGGGGCCACCTCCC TCAGAGGCTCTGCTAGCCACACTCCGTGGCCCACCCTTTG TTACCAGTTCTTCCTCCTTCCTCTTTTCCCCTGCCTTTCT CATTCCTTCCTTCGTCTCCCTTTTTGTTCCTTTGCCTCTT GCCTGTCCCCTAAAACTTGACTGTGGCACTCAGGGTCAAA CAGACTATCCATTCCCCAGCATGAATGTGCCTTTTAATTA GTGATCTAGAAAGAAGTTCAGCCGAACCCACACCCCAACT CCCTCCCAAGAACTTCGGTGCCTAAAGCCTCCTGTTCCAC CTCAGGTTTTCACAGGTGCTCCCACCCCAGTTGAGGCTCC CACCCACAGGGCTGTCTGTCACAAACCCACCTCTGTTGGG AGCTATTGAGCCACCTGGGATGAGATGACACAAGGCACTC CTACCACTGAGCGCCTTTGCCAGGTCCAGCCTGGGCTCAG GTTCCAAGACTCAGCTGCCTAATCCCAGGGTTGAGCCTTG TGCTCGTGGCGGACCCCAAACCACTGCCCTCCTGGGTACC AGCCCTCAGTGTGGAGGCTGAGCTGGTGCCTGGCCCCAGT CTTATCTGTGCCTTTACTGCTTTGCGCATCTCAGATGCTA ACTTGGTTCTTTTTCCAGAAGCCTTTGTATTGGTTAAAAA TTATTTTCCATTGCAGAAGCAGCTGGACTATGCAAAAAGT ATTTCTCTGTCAGTTCCCCACTCTATACCAAGGATATTAT TAAAACTAGAAATGACTGCATTGAGAGGGAGTTGTGGGAA ATAAGAAGAATGAAAGCCTCTCTTTCTGTCCGCAGATCCT GACTTTTCCAAAGTGCCTTAAAAGAAATCAGACAAATGCC CTGAGTGGTAACTTCTGTGTTATTTTACTCTTAAAACCAA ACTCTACCTTTTCTTGTTGTTTTTTTTTTTTTTTTTTTTT TTTTTTTGGTTACCTTCTCATTCATGTCAAGTATGTGGTT CATTCTTAGAACCAAGGGAAATACTGCTCCCCCCATTTGC TGACGTAGTGCTCTCATGGGCTCACCTGGGCCCAAGGCAC AGCCAGGGCACAGTTAGGCCTGGATGTTTGCCTGGTCCGT GAGATGCCGCGGGTCCTGTTTCCTTACTGGGGATTTCAGG GCTGGGGGTTCAGGGAGCATTTCCTTTTCCTGGGAGTTAT GACCGCGAAGTTGTCATGTGCCGTGCCCTTTTCTGTTTCT GTGTATCCTATTGCTGGTGACTCTGTGTGAACTGGCCTTT GGGAAAGATCAGAGAGGGCAGAGGTGGCACAGGACAGTAA AGGAGATGCTGTGCTGGCCTTCAGCCTGGACAGGGTCTCT GCTGACTGCCAGGGGCGGGGGCTCTGCATAGCCAGGATGA CGGCTTTCATGTCCCAGAGACCTGTTGTGCTGTGTATTTT GATTTCCTGTGTATGCAAATGTGTGTATTTACCATTGTGT AGGGGGCTGTGTCTGATCTTGGTGTTCAAAACAGAACTGT ATTTTTGCCTTTAAAATTAAATAATATAACGTGAATAAAT GACCCTATCTTTGTAA

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