Compositions And Methods For Reducing Fucosylation Of Glycoproteins In Insect Cells And Methods Of Use Thereof For Production Of Recombinant Glycoproteins

Abstract

Compositions for reducing fucosylation of glycoproteins in insect cells are provided. Also disclosed are methods of use of such compositions for the production of recombinant humanized proteins.

Description

PRIORITY CLAIM

[0001] This application claims priority to U.S. Provisional Application Number 61/885,294 filed Oct. 1, 2013. This application is incorporated herein by reference as though set forth in full.

FIELD OF THE INVENTION

[0003] This invention relates to the fields of molecular biology and production of glycoproteins lacking fucosylation. More specifically, the invention provides compositions and methods for expressing a GDP-4-dehydro-6-deoxy-D-mannose reductase enzyme encoded by a recombinant baculovirus vector that blocks the production of GDP-L-fucose and generates a molecule lacking or having a reduced amount of fucose.

BACKGROUND OF THE INVENTION

[0004] Numerous publications and patent documents, including both published applications and issued patents, are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.

[0005] In view of bioinformatic analyses suggesting that well over half of all human proteins are glycosylated, the ability to support glycosylation is an increasingly significant attribute of recombinant protein production systems, including insect-based systems, such as the baculovirus-insect cell system (reviewed by Jarvis 2009; Usami et al. 2010). The native human protein glycosylation process often results in the addition of terminally sialylated N-glycans that dramatically influence key properties, such as the in vivo half-lives, of glycoproteins in the human body (Ngantung et al. 2006; reviewed by Varki and Gagneux 2012). Most investigators know that insect-based systems, including the baculovirus-insect cell expression system, support recombinant protein glycosylation, but it is also important to recognize that these systems cannot produce human-type, terminally sialylated N-glycans (Marchal et al. 2001; Hillar and Jarvis 2010). This limitation has been addressed by glycoengineering baculovirus vectors and/or insect cell lines to encode and express mammalian glycogenes that extend endogenous insect cell N-glycan processing capabilities (Jarvis and Finn 1996; Wagner et al. 1996; Hollister et al. 1998; Ailor et al. 2000; Seo et al. 2001; Jarvis et al. 2001; Hollister and Jarvis 2001; Hollister et al. 2002; Aumiller et al. 2003; Tomiya et al. 2003; Chang et al. 2003; Yun et al. 2005; Hill et al. 2006; Okada et al. 2010; Aumiller et al. 2012; Geisler and Jarvis 2012; Palmberger et al. 2012; Mabashi-Asazuma et al. 2013). These efforts ultimately yielded new baculovirus-insect systems that can produce recombinant glycoproteins with human-type, terminally sialylated N-glycans. This was an important step towards the broader goal of extending the utility of the baculovirus-insect cell system to include therapeutic glycoprotein production, which is not currently considered to be a legitimate application of this system. In order to finally achieve this goal, however, it also will be necessary to eliminate core .alpha.1,3-fucosylation of recombinant glycoproteins in certain insect cell lines, including some that are commonly used as hosts for baculovirus vectors, as this modification generates a highly immunogenic carbohydrate epitope (reviewed by Fotisch and Vieths 2001; Altmann et al. 2007).

[0006] There are two distinct types of N-glycan core fucosylation involving the addition of either .alpha.1,6- or .alpha.1,3-linked fucose residues to the core N-acetylglucosamine. Humans encode a single core fucosyltransferase, FUT8, which can only add .alpha.1,6-linked fucose residues, but many insects encode two core fucosyltransferases, FucT6 and FucTA, which can add .alpha.1,6- or .alpha.1,3-linked fucose residues, respectively, to the core N-acetylglucosamine (FIG. 1A; Fabini et al. 2001; Paschinger et al. 2005; Rendi et al. 2007). Because humans have no FucTA counterpart, we cannot produce core .alpha.1,3-fucosylated N-glycoproteins and, as a result, the core .alpha.1,3-fucosylated sugar epitope is immunogenic for many people. In fact, the presence of this epitope on bee venom glycoproteins accounts for the life-threatening allergic responses, such as anaphylactic shock, induced by bee stings in some humans (King et al. 1976; Prenner et al. 1992; Kubelka et al. 1995; reviewed by Altmann et al. 2007). In addition, pre-existing human antibodies against the immunogenic sugar epitope can give false positive results in diagnostic tests that use .alpha.1,3-fucosylated recombinant glycoproteins produced in the baculovirus-insect cell system (Hancock et al. 2008; Seismann et al. 2010).

[0007] Among the insect cell lines commonly used as hosts for baculovirus-mediated recombinant protein production, BTI-Tn-5B1-4 (Wickham et al. 1992; commercialized as High Five.TM. by Life Technologies Inc., Carlsbad, Calif.), which is derived from Trichoplusia ni, produces high levels of the core .alpha.1,3-fucosylated N-glycan epitope, whereas Sf9 (Summers and Smith 1987), derived from Spodoptera frugiperda, and BmN (Maeda 1989), derived from Bombyx mori, do not (Rudd et al. 2000; Hancock et al. 2008; Seismann et al. 2010; Blank et al. 2011; Palmberger et al. 2011). Importantly, the capacity to produce immunogenic, core .alpha.1,3-fucosylated N-glycans trumps the opportunity to exploit the potentially higher recombinant glycoprotein production capacity of insect cell lines derived from Trichoplusia ni, such as High Five.TM. (Davis et al. 1992; Krammer et al. 2010).

[0008] While core .alpha.1,3-fucosylation is a relatively host-specific modification, core .alpha.1,6-fucosylation is a common feature of recombinant glycoproteins produced by all insect cell lines, including those used as hosts for baculovirus vectors. As noted above, core .alpha.1,6-fucosylation also occurs in humans and, therefore, does not produce an immunogenic sugar epitope. Nevertheless, this form of core fucosylation is also biotechnologically significant because it inhibits the effector functions of certain types of therapeutic antibodies (Shields et al. 2002; Shinkawa et al. 2003; reviewed by Satoh et al. 2006; Jefferis 2009), which comprise a large and growing share of the human biologics market (reviewed by Elvin et al. 2013). The inhibition of antibody effector functions by core fucosylation stimulated efforts to block pathways responsible for this modification and enable production of non-fucosylated antibodies in mammalian cell expression systems (FIG. 1B). Various approaches included repressing or eliminating FUT8 gene expression (Yamane-Ohnuki et al. 2004; Imai-Nishiya et al. 2007; Malphettes et al. 2010), overexpressing upstream processing enzymes to produce N-glycan structures that are not recognized as acceptor substrates (Ferrara et al. 2006; Zhong et al. 2012), and blocking production of GDP-L-fucose, which is required as the donor substrate for fucosylation by FUT8 (Imai-Nishiya et al. 2007; Kanda et al. 2007; von Horsten et al. 2010). To date, however, there have been no reported efforts to block recombinant glycoprotein fucosylation in any insect-based system, including the baculovirus-insect cell system, despite the arguably more serious problem of immunogenicity associated with core .alpha.1,3-fucosylation mediated by some insect and insect cell types.

SUMMARY OF THE INVENTION

[0009] In accordance with the present invention, a recombinant baculovirus expression vector for transient expression of GDP-4-dehydro-6-deoxy-D-mannose reductase (RMD) in an insect cell is disclosed. In one embodiment, the vector comprises the following operably linked components, i) an expression control sequence functional early in infection operably linked to a codon optimized RMD encoding nucleic acid; and ii) an insertion site suitable for insertion of one or more nucleic acids encoding at least one heterologous protein of interest. In a particularly preferred embodiment, the vector is AcRMD shown in FIG. 14. In another embodiment, the vector further comprises a nucleic acid sequence encoding at least one heterologous protein of interest operably linked to a promoter which is active later in infection, e.g., a p6.9 or polyhedrin promoter inserted at said insertion site.

[0010] In certain embodiments, the expression control sequence includes a promoter selected from the group consisting of baculovirus immediate early and delayed early promoters and an inducible promoter. In a preferred embodiment, the expression control sequence also includes an enhancer element.

[0011] The vectors of the invention have utility in the production of non-fucosylated therapeutic proteins. Such therapeutic proteins include, without limitation, an antibody, a subunit vaccine, an antibiotic, a cytokine, an anticoagulant, a viral antigen, an enzyme, a hormone, or a blood clotting factor. Cells useful in the methods disclosed herein include, for example, Sf9, Sf21, expresSF+.RTM., Tn368, High Five.RTM., Tni PRO.RTM., Ea4, Ao38, BmN, S2, and S2R+ cells.

[0012] Also provided is a method for producing at least one molecule of interest (e.g., a therapeutic protein) lacking fucose, comprising providing insect cells and introducing a baculovirus comprising at least one nucleic acid molecule encoding a codon optimized enzyme GDP-4-dehydro-6-deoxy-D-mannose reductase (RMD) operably driven by an immediate early expression control sequence for expression immediately after infection or an inducible promoter, and at least one additional nucleic acid encoding a protein of interest driven by a promoter active later in infection that may or may not be on the same vector encoding RMD. Using this approach, inhibition of fucosylation is stabilized permitting production of a non-fucosylated protein of interest. The infected cells are incubated under conditions wherein GDP-4-dehydro-6-deoxy-D-mannose reductase blocks the production of GDP-L-fucose, and said at least one protein of interest is produced lacking fucose. Following production, the non-fucosylated protein of interest is isolated.

[0013] In yet another aspect, a kit for the production of at least one protein of interest lacking fucose is provided comprising at least one recombinant baculovirus comprising at least one nucleic acid molecule encoding the codon optimized enzyme GDP-4-dehydro-6-deoxy-D-mannose reductase (RMD) operably driven by an immediate early expression control sequence for expression immediately after infection, thereby stabilizing inhibition of fucosylation, and an insertion site for at least one additional nucleic acid molecule encoding at least one protein of interest, for production of non-fucosylated proteins of interest. The kit may also contain insect cells and at least one additional baculoviral vector comprising a promoter suitable to drive expression of the protein of interest and an insertion site for the at least one nucleic acid encoding the protein of interest.

[0014] In a further embodiment, the invention provides a method for production of a non-fucosylated protein in insect larvae. An exemplary method entails providing insect larvae and introducing therein a baculovirus comprising at least one nucleic acid molecule encoding a codon optimized enzyme GDP-4-dehydro-6-deoxy-D-mannose reductase (RMD) operably driven by an immediate early expression control sequence for expression immediately after infection or an inducible promoter, thereby stabilizing inhibition of fucosylation, and at least one additional nucleic acid molecule encoding at least one protein of interest driven by an promoter active later in infection, thereby producing non-fucosylated proteins wherein said additional nucleic acid is present on the same baculovirus encoding RMD or is present on a second baculovirus vector. The infected larvae are then incubated under conditions wherein GDP-4-dehydro-6-deoxy-D-mannose reductase blocks the production of GDP-L-fucose, and said at least one protein of interest is produced lacking fucose. The method further includes the step of isolating the protein of interest.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1. Pathways of N-glycan fucosylation (A) and GDP-L-fucose biosynthesis (B). In theory, GDP-L-fucose biosynthesis in eukaryotic cells can be blocked by expression of bacterial RMD, which converts GDP-4-keto-6-deoxy-D-mannose to GDP-D-rhamnose. Abbreviations: FUT8, human core .alpha.1,6-fucosyltransferase 8; FucT6, insect core .alpha.1,6-fucosyltransferase; FucTA, insect core .alpha.1,3-fucosyltransferase; GMD, GDP-D-mannose 4,6-dehydratase; Fx protein, GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase/4-reductase; GFR, Golgi GDP-L-fucose transporter; Rmd, GDP-4-dehydro-6-deoxy-D-mannose reductase; FUK, fucokinase; FPGT, fucose-1-phosphate guanylyltransferase.

[0016] FIG. 2. Core .alpha.1,3-fucosylation of endogenous insect cell glycoproteins. Total proteins in Sf9, High Five.TM., or Tni PRO.TM. cell lysates were resolved by SDS-PAGE in 12% acrylamide gels and stained with Coomassie Brilliant Blue (A) or transferred to a PVDF membrane and analyzed by western blotting with primary anti-HRP rabbit IgG and secondary a-rabbit IgG conjugated to alkaline phosphatase (B).

[0017] FIG. 3. Phylogenetic trees of genes involved in GDP-L-fucose biosynthesis. Phylogenetic analysis of FUK (A), FPGT (B), GMD (C), and Fx protein (D) genes was performed using the Maximum Likelihood method with MEGA5 software (Tamura et al., 2011). The bars indicate the number of substitutions per site. Abbreviations: Hs, Homo sapiens; Mm, Mus musculus; Bt, Bos taurus; Gg, Gallus gallus; X1, Xenopus laevis; Dr, Danio rerio; Dp, Daphnia pulex; Ce, Caenorhabditis elegans; At, Arabidopsis thaliana.

[0018] FIG. 4. Cell surface fucosylation. Sf9 and polyclonal SfRMD (A) or High Five.TM. and polyclonal TnRMD (B) cells were seeded into culture plates, and then stained with AAL, as described in Materials and methods. The cell surface staining patterns are shown alongside corresponding phase contrast micrographs, as indicated.

[0019] FIG. 5. Sf9, SfRMD 2B2, High Five.TM. and TnRMD 6A6 cells were infected with a baculovirus vector encoding a 6X HIS-tagged Fc domain of mouse IgG2a (mIgG2a-Fc) under the control of the baculovirus p6.9 promoter and the mIgG2a-Fc was affinity-purified from the extracellular fractions, as described in Materials and methods. Samples were then treated with peptide-N.sup.4-(N-acetyl-.beta.-glucosaminyl) asparagine amidase (PNGase)-F or reaction buffer alone, resolved by SDS-PAGE, transferred to a PVDF membrane, and probed with AAL to detect fucose or with anti-mouse IgG to detect the protein.

[0020] FIG. 6. Impact of AcP(+)IE1-RMD co-infection on mIgG2a-Fc fucosylation. Sf9 or Tni PRO.TM. cells were infected with Acp6.9-mIgG2a-Fc, a recombinant baculovirus encoding mIgG2a-Fc, or with equal doses of Acp6.9-mIgG2a-Fc and AcP(+)IE1-RMD, a recombinant baculovirus encoding Rmd. The cell-free media were harvested at 48 hours post-infection and used to affinity purify each mIgG2a-Fc preparation for lectin blotting analysis with AAL (specific for fucose) or western blotting analysis with anti-HRP (specific for core .alpha.1,3 fucose) or anti-mouse IgG with (+) or without (-) PNGase-F pre-treatment, as indicated.

[0021] FIG. 7. N-glycan profiling of various mIgG2a-Fc preparations by MALDI-TOF MS. mIgG2a-Fc preparations were produced and purified from Sf9 and Tni PRO.TM. cells infected with Acp6.9-mIgG2a-Fc alone or Acp6.9-mIgG2a-Fc and AcP(+)IE1-RMD, as described in the legend to FIG. 6. PNGaseAr was used to remove the N-glycans, which were then recovered, permethylated, and analyzed by MALDI-TOF MS, as described in Materials and methods. mIgG2a-Fc from Sf9 cells infected with Acp6.9-mIgG2a-Fc alone (A), Sf9 cells co-infected with Acp6.9-mIgG2a-Fc and AcP(+)IE1-RMD (B), Tni PRO.TM. cells infected with Acp6.9-mIgG2a-Fc alone (C), or Tni PRO.TM. cells co-infected with Acp6.9-mIgG2a-Fc and AcP(+)IE1-RMD (D). All molecular ions were detected as [M+Na].sup.+, assigned, and annotated using the standard cartoon symbolic representations.

[0022] FIG. 8. Genetic maps of parental baculoviral vector, baculovirus transfer plasmid, and AcRMD. The new baculovirus vector designated AcRMD was isolated by replacing the 5' regions of the AcMNPV chiA and v-cath genes in BacPAK6 (Kitts and Possee 1993) baculoviral DNA with an expression cassette encoding EGFP and Rmd under the control of dual, back-to-back ie1 promoters separated by the AcMNPV hr5 enhancer, as described in Materials and methods.

[0023] FIG. 9. Genetic maps of recombinant baculoviruses encoding anti-CD20-IgG. The recombinant baculoviruses designated Ac-.alpha.CD20-IgG and Acp6.9-.alpha.CD20-IgG were isolated by using homologous recombination to replace the lacZ sequence in the parental baculovirus, AcGT (Toth et al, 2011), with expression cassettes encoding the anti-CD20-IgG heavy and light chains under the control of dual, back-to-back polyhedrin or p6.9 promoters separated by the second intron of the Drosophila melanogaster white gene, respectively. Similarly, the recombinant baculoviruses designated AcRMD-.alpha.CD20-IgG and AcRMDp6.9-.alpha.CD20-IgG were isolated by using homologous recombination to replace the lacZ sequence of the parental baculovirus, AcRMD (this study), with the same expression cassettes used to isolate Ac-.alpha.CD20-IgG and Acp6.9-.alpha.CD20-IgG.

[0024] FIG. 10. Expression and secretion of anti-CD20-IgG under the control of late or very late baculoviral promoters. Sf9 cells were infected with AcMNPV, Ac-CD20-IgG, Acp6.9-.alpha.CD20-IgG, AcRMD-.alpha.CD20-IgG, or AcRMDp6.9-.alpha.CD20-IgG. At 48 hours post-infection, the cell-free culture media were collected as the extracellular fraction, the cells were lysed, and the clarified supernatants were collected as the intracellular fraction. Proteins were resolved by SDS-PAGE, transferred to a PVDF membrane, and probed with anti-human IgG Fc-specific (.alpha.-HC) or anti-human IgG .kappa. chain-specific antibodies (.alpha.-LC).

[0025] FIG. 11. Analysis of purified anti-CD20-IgG preparations from Sf9 and Tni PRO.TM.. The anti-CD20-IgG preparations produced by Sf9 or Tni PRO.TM. cells infected with Acp6.9-.alpha.CD20-IgG or AcRMDp6.9-.alpha.CD20-IgG were affinity purified using protein A, as described in Materials and methods, resolved by SDS-PAGE under reducing (A) or non-reducing (B) conditions, and stained with Coomassie Brilliant Blue.

[0026] FIG. 12. Fucosylation of an anti-CD20-IgG (rituximab) produced using baculovirus or AcRMD baculovirus vectors. Sf9 and Tni PRO.TM. cells were infected with either Acp6.9-.alpha.CD20-IgG or AcRMDp6.9-.alpha.CD20-IgG, and the cell-free media were harvested and used to affinity purify anti-CD20-IgG at 48 hours post-infection. The results of western blotting assays with anti-human IgG Fc (A, .alpha.-HC) or anti-human IgG .kappa. chain (B, .alpha.-LC) and lectin blotting assays with AAL (C) with (+) or without (-) PNGase-F pre-treatment are shown.

[0027] FIG. 13. N-glycan profiling of various anti-CD20-IgG preparations by MALDI-TOF MS. Anti-CD20-IgG was isolated from Sf9 or Tni PRO.TM. cells infected with Acp6.9-.alpha.CD20-IgG or AcRMDp6.9-.alpha.CD20-IgG, as described in the legend of FIG. 12. The N-glycans were removed by PNGaseAr treatment, recovered, permethylated, and analyzed by MALDI-TOF MS, as described in Materials and methods. Anti-CD20-IgG from Sf9 cells infected with Acp6.9-.alpha.CD20-IgG (A), Sf9 cells infected with AcRMDp6.9-.alpha.CD20-IgG (B), Tni PRO.TM. cells infected with Acp6.9-.alpha.CD20-IgG (C), or Tni PRO.TM. cells infected with AcRMDp6.9-.alpha.CD20-IgG (D). All molecular ions were detected as [M+Na].sup.+, assigned, and annotated using the standard cartoon symbolic representations.

[0028] FIG. 14. A plasmid map (FIG. 14A) showing the major genetic elements and text files (FIGS. 14B to 14E) of the hr5 enhancer, AcMNPV ie1 promoter, codon-optimized RMD encoding sequence and the optional EGFP encoding sequences in the transfer plasmid p.DELTA.Chi/Cath-EGFP/RMD, which was used to isolate AcRMD (SEQ ID NO: 1). In certain embodiments the EGFP encoding sequences are removed from SEQ ID NO: 1.

[0029] FIG. 15. FIGS. 15A-15D show the pVL1393-polh-antiCD20-IgG sequence containing the AcMNPV polyhedrin promoter, anti-CD20-IgG heavy chain coding sequence, and anti-CD20-IgG light chain coding sequence (SEQ ID NO: 2), which was used to isolate a daughter of AcRMD encoding anti-CD20 under the control of the polyhedrin promoter.

[0030] FIG. 16. FIGS. 16A-16D show text files of pVL1393-p6.9-antiCD20-IgG sequence containing the AcMNPV p6.9 promoter, anti-CD20-IgG heavy chain coding sequence, and anti-CD20-IgG light chain coding sequence (SEQ ID NO: 3), which was used to isolate a daughter of AcRMD encoding anti-CD20 under the control of the p6.9 promoter.

DETAILED DESCRIPTION OF THE INVENTION

[0031] The ability to glycosylate recombinant proteins is an important attribute of insect-based, including baculovirus-insect cell expression systems, but some insect cell lines produce recombinant glycoproteins with core .alpha.1,3-fucosylated N-glycans, which are highly immunogenic and render products unsuitable for human use. To address this problem, we exploited a bacterial enzyme, GDP-4-dehydro-6-deoxy-D-mannose reductase (Rmd), which consumes the precursor to GDP-L-fucose. We expected this enzyme to indirectly block glycoprotein fucosylation by blocking the production of GDP-L-fucose, which is required as the donor substrate for this process. Initially, we genetically transformed two different insect cell lines to constitutively express Rmd and successfully isolated subclones with fucosylation-negative phenotypes. Surprisingly, however, we found that the fucosylation-negative phenotypes induced by Rmd expression were unstable, indicating that the prior art involving host cell engineering is ineffective in insect systems. Thus, we constructed a novel baculovirus vector designed to express Rmd immediately after infection and to facilitate the insertion of genes encoding any glycoprotein of interest for expression at a later time after infection. We used this vector to produce a daughter encoding a therapeutic anti-CD20-IgG (rituximab) and found, in contrast to an Rmd-negative control, that insect cells infected with this virus produced a non-fucosylated form of this antibody. These results indicate that the novel Rmd.sup.+ baculoviral vector we produced can be used to solve the problem of immunogenic core .alpha.1,3-fucosylated N-glycan production associated with insect cell systems, including the baculovirus-insect cell system. These results extend the utility of such systems, which, when used in conjunction with existing glycoengineered insect cell lines, now include therapeutic glycoprotein production, in particular, production of recombinant antibodies lacking fucose with enhanced effector functions.

DEFINITIONS

[0032] A "cell line" refers to cells that can be cultured in the lab for an indefinite period and are useful for producing large amounts of a protein of interest.

[0033] As used herein, the term "insect" includes an insect in any stage of development, including first through fifth instar larvae. For the production of non-fucosylated polypeptides of interest, a large larva, such as a third, fourth, or fifth instar larva is preferred. It will be evident to a skilled worker which insect stage is suitable for a particular purpose, such as for direct production of a glycosylated polypeptide of interest, for storage or transport of an insect to a different location, for generation of progeny, for further genetic crosses, or the like.

[0034] With reference to nucleic acids of the invention, the term "isolated nucleic acid" is sometimes used. This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous (in the 5' and 3' direction) in the naturally occurring genome of the organism from which it originates. For example, the "isolated nucleic acid" may comprise a DNA or cDNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the DNA of a prokaryote or eukaryote. Isolated nucleic acids refers to those suitable for insertion in the recombinant baculoviral vectors described herein, e.g., RMD encoding nucleic acids and those encoding the desired protein of interest.

[0035] With respect to protein, the term "isolated protein" or "isolated and purified protein" is sometimes used herein. This term refers primarily to a protein produced by expression of an isolated nucleic acid molecule of the invention. Alternatively, this term may refer to a protein that has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in "substantially pure" form.

[0036] The term "protein of interest" is sometimes used herein. The term refers to any protein and includes, without limitation, therapeutic proteins, antibodies, a subunit vaccine, an antibiotic, a cytokine, an anticoagulant, a viral antigen, an enzyme, a hormone, or a blood clotting factor.

[0037] The term "promoter region or expression control sequence" refers to the transcriptional regulatory regions of a gene, which may be found at the 5' or 3' side of the coding region, or within the coding region, or within introns. Such sequences regulate expression of a polypeptide coded for by a polynucleotide to which it is functionally ("operably") linked. Expression can be regulated at the level of the mRNA or polypeptide. Thus, the term expression control sequence includes mRNA-related elements and protein-related elements. Such elements include promoters, domains within promoters, upstream elements, enhancers, elements that confer tissue or cell specificity, response elements, ribosome binding sequences, transcriptional terminators, etc.

[0038] Suitable expression control sequences that can function in insect cells will be evident to the skilled worker. In some embodiments, the coding sequences described herein may be operably linked to an expression control sequence from the virus, itself, or to another suitable expression control sequence. Suitable baculovirus vectors include those based on Autographa californica NPV, Orgyia pseudotsugata NPV, Lymantria dispar NPV, Bombyx mori NPV, Rachoplusia ou NPV, Spodoptera exigua NPV, Heliothis zea NPV, Galleria mellonella NPV, Anagrapha falcifera nucleopolyhedrovirus (AfNPV), Trichoplusia ni singlenuclepolyhedrovirus (TnSNPV). As discussed above, baculovirus-based vectors have been generated (or can be generated without undue experimentation) that allow the cloning of large numbers of inserts, at any of a variety of cloning sites in the viral vector. Thus, more than one heterologous polypeptide may be introduced together into a transgenic insect cell or insect of the invention. The viral vector can be introduced into an insect cell or insect by known methods, such as by in vitro inoculation (insect cells) or injection or oral ingestion (insect larvae). Among the many suitable "strong" promoters that can be used are the baculovirus p10, polyhedrin (polh), p6.9, capsid, and v-cath promoters. Among the many suitable "weak" promoters are the baculovirus ie1, ie2, ie0, et1, 39K (aka pp31), and gp64 promoters. Other suitable constitutive promoters include insect actin hsp70, .alpha.1-tubulin, and ubiquitin gene promoters; RSV and MMTV promoters, copia promoter, gypsy promoter, and the cytomegalovirus IE promoter. If it is desired to increase the amount of gene expression from a weak promoter, enhancer elements, such as the baculovirus enhancer elements, hr5 or hr2, among others, may be used in conjunction with the promoter.

[0039] In some embodiments of the invention, as is discussed in more detail elsewhere herein, it is desirable that an expression control sequence is regulatable (e. g., comprises an inducible promoter and/or enhancer element). Suitable regulatable promoters include, e.g., hsp70 promoters, the Drosophila metallothionein promoter, an insect ecdysone-regulated promoter, the Saccharomyces cerevisiae Gal4/UAS system, and other well-known inducible promoter systems. A Tet-regulatable molecular switch may be used in conjunction with any constitutive promoter, such as those described elsewhere herein (e. g, in conjunction with the CMV-IE promoter, or baculovirus promoters). Another type of inducible promoter is a baculovirus delayed early, late or very late promoter that is only activated following infection by a baculovirus.

[0040] In some embodiments, the expression control sequence comprises a tissue-or organ-specific promoter. Many such expression control sequences will be evident to the skilled worker. The term "vector" refers to a small carrier DNA molecule into which a DNA sequence can be inserted for introduction into a host cell where it will be replicated. An "expression vector" is a specialized vector that contains a gene or nucleic acid sequence with the necessary regulatory regions needed for expression in a host cell. A "baculovirus expression vector" is an expression vector consisting of a recombinant baculovirus that contains a gene or nucleic acid sequence with the necessary regulatory regions needed for expression in a host cell.

[0041] The term "operably linked" means that the regulatory sequences necessary for expression of a coding sequence are placed in the DNA molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of coding sequences and transcription control elements (e.g. promoters, enhancers, and termination elements) in an expression vector. This definition is also sometimes applied to the arrangement of nucleic acid sequences of a first and a second nucleic acid molecule wherein a hybrid nucleic acid molecule is generated.

[0042] The phrase "consisting essentially" of when referring to a particular nucleotide sequence or amino acid sequence means a sequence having the properties of a given SEQ ID NO:. For example, when used in reference to an amino acid sequence, the phrase includes the sequence per se and molecular modifications that would not affect the basic and novel characteristics of the sequence.

[0043] Methods for designing and preparing constructs suitable for generating recombinant baculovirus vectors for infection of insect cells or insects are known to one of ordinary skill in the art. For these methods, as well as other molecular biology procedures related to the invention, see, e. g., Sambrook et al. 1989; Wu et al. 1997; and Ausabel et al. 1994-1999.

[0044] In a preferred embodiment, the baculovirus replicates until the host insect cell is killed. The insect cell or insect lives long enough to produce large amounts of the non-fucosylated polypeptide of interest. In another embodiment, a baculovirus is used that is attenuated or non-permissive for the host. In this case, the host is not killed by replication of the baculovirus, itself (although the host may be damaged by expression of the heterologous protein of interest).

[0045] The following materials and methods are provided to facilitate the practice of the present invention.

Plasmid Constructions

[0046] The Pseudomonas aeruginosa Rmd (GenBank: AAG08839.1) coding sequence was optimized for Spodoptera frugiperda, synthesized by GeneArt.RTM. (Life Technologies, Gaithersburg, Md.), and cloned into pMK-T to produce a plasmid designated pKan-RMD.

[0047] pIE1-RMD, a plasmid designed to express Rmd under the control of the AcMNPV ie1 promoter, was constructed by subcloning the BamHI-NotI fragment of pKan-RMD into the BamHI and NotI sites of pIE1TV4 (Jarvis et al. 1996).

[0048] pAcP(+)IE1-RMD, a baculovirus transfer plasmid, was constructed by subcloning the BamHI-NotI fragment of pIE1-RMD into the BglII and NotI sites of pAcP(+)IE1TV3 (Jarvis et al. 1996).

[0049] pDIE1-EGFP/RMD.DELTA.Bsu36I, a plasmid designed to express both EGFP and Rmd under the control of dual, back-to-back AcMNPV ie1 promoters, was constructed in four sequential steps. First, the bovine growth hormone (BGH) polyadenylation (poly A) signal was amplified by polymerase chain reaction (PCR) with pcDNA3.1 (Life Technologies) as the template and PmeI-BGHpolyA-Fw and SpeI-BGHpolyA-Rv as the primers. The resulting amplimer was digested with PmeI and SpeI and cloned into the corresponding sites of pIE1TV4 to produce a plasmid designated pIE1-BR. Second, the EGFP coding sequence was amplified by PCR with pIE1-EGFP as the template and BamHI/KpnI-EGFP-Fw and NruI-EGFPstop-Rv as the primers. The resulting amplimer was digested with BamHI and NruI and subcloned into the BamHI and PmeI sites of pIE1-BR to produce a plasmid designated pIE1-EGFP-BGH. Third, the Bsu36I site of Rmd was deleted by overlapping PCR mutagenesis. This involved amplifying the 5'-region of Rmd with NruI-RMD-Fw and RMD-BsuDe1-Rv as the primers and the 3'-region of Rmd with RMD-BsuDe1-Fw and ApaI-RMD-Rv as the primers, with pIE1-RMD as the template in both cases. The resulting 5'- and 3'-region Rmd amplimers were mixed and used as templates for an overlapping PCR with NruI-RMD-Fw and ApaI-RMD-Rv as the primers. The resulting RMDBsu36I amplimer was subcloned into pGEM-T (Promega, Madison, Wis.) to produce pDIE1-EGFP/RMD.DELTA.Bsu36I. Finally, the EGFP-BGH poly A signal and RMD.DELTA.Bsu36I coding sequence were assembled by sequentially subcloning the KpnI-HindIII fragment of pIE1-EGFP-BGH and the NruI-ApaI fragment of pGEMT-RMD.DELTA.Bsu36I into the corresponding sites of pDIE1-TOPO.3 (Shi et al. 2007) to produce the plasmid designated pDIE1-EGFP/RMD.DELTA.Bsu36I.

[0050] To construct a baculovirus transfer vector targeting the chiA/v-cath locus, we produced three PCR amplimers comprised of AcMNPV orf124/lef7, chiA, or v-cath/gp64 sequences and independently subcloned each one into pGEM-T. After verifying their sequences, these three DNA fragments were assembled by sequentially subcloning the PmeI-ApaI fragment of chiA and then the ApaI fragment of v-cath/gp64 into the corresponding sites of pGEMT-orf124/lef7 to produce a new plasmid designated pChi/CathTVC6. Finally, we blunt-ended the HindIII-BamHI fragment of pDIE1-EGFP/RMD.DELTA.Bsu36I with T4 DNA polymerase (New England Biolabs, Beverly, Mass.) and inserted the resulting DNA fragment into the T4 DNA polymerase blunt-ended HindIII and EcoRI sites of pChi/CathTVC6. This transfer plasmid, which was designated p.DELTA.Chi/Cath-EGFP/RMD.DELTA.Bsu36I, was used to create the new baculovirus vector in which the viral chiA and v-cath genes were replaced by genes encoding Rmd and EGFP, each under the control of the AcMNPV ie1 promoter.

[0051] pVL1393-polh-.alpha.CD20-IgG and pVL1393-p6.9-.alpha.CD20-IgG, which are baculovirus transfer plasmids used to isolate baculovirus expression vectors encoding anti-CD20-IgG, were constructed in four sequential steps. First, the BGH poly A signal was PCR amplified with pIE 1-BR as the template and EcoRI-BGHpolyA-Fw and EcoRV-BGHpolyA-Rv as the primers. The resulting amplimer was digested with EcoRI and EcoRV and then subcloned into the corresponding sites of pVL1393 (Summers and Smith 1987) to produce pVL1393-BGH. Second, the NotI-SbfI fragment of pGEMT-.alpha.CD20HC, which encodes the heavy chain, and the NotI-EcoRI fragment of pGEMT-.alpha.CD20LC, which encodes the light chain of anti-CD20-IgG were sequentially subcloned into the NotI-PstI and NotI-EcoRI sites of pVL1393-BGH to produce pVL1393-.alpha.CD20-IgG-no promoter. Third, two copies of the AcMNPV polyhedrin or p6.9 promoter were assembled in back-to-back orientation, with the second intron of the Drosophila melanogaster white gene as an intervening spacer, to create plasmids designated either pGEMT-ppol-wi-ppol or pGEMT-p6.9-wi-p6.9, respectively. The former was constructed by PCR-amplifying two copies of the polyhedrin promoter with pAcGT N-term 8xHis pPol (Toth et al. 2011) as the template and either NheI-ppol-Fw and NotI-ppol-Rv or XhoI-ppol-Fw and SphI/PmeI-ppol-Rv as the primers. The resulting amplimers were digested with NheI and NotI or XhoI and SphI, respectively, and sequentially subcloned into the corresponding sites of pGEM-WIZ (Bao and Cagan 2006). Similarly, pGEMT-p6.9-wi-p6.9 was constructed by PCR-amplifying two copies of the p6.9 promoter with pAcp6.9GT N-term 8xHis pPol (Toth et al. 2011) as the template and either NheI-p6.9-Fw and NotI-p6.9-Rv or XhoI-p6.9-Fw and SphI/PmeI-p6.9-Rv as the primers. The resulting amplimers were digested with NheI and NotI or XhoI and SphI, respectively, and sequentially subcloned into the corresponding sites of pGEM-WIZ. Finally, each back-to-back promoter cassette was independently subcloned into the anti-CD20-IgG baculovirus transfer plasmid described above. pVL1393-polh-.alpha.CD20-IgG was constructed by subcloning the PmeI-NotI fragment of pGEMT-ppol-wi-ppol and pVL1393-p6.9-.alpha.CD20-IgG was constructed by subcloning the PmeI-NotI fragment of pGEMT-p6.9-wi-p6.9 into the corresponding sites of pVL1393-.alpha.CD20-IgG_no promoter, respectively.

[0052] Phusion.RTM. Taq DNA polymerase (New England Biolabs) and a Biometra TProfessional Standard Thermocycler (Gottingen, Germany) were used for all PCRs and all primer sequences are given in Table 1.

TABLE-US-00001 TABLE 1 Primers used in this study. Primer name Sequence (5' to 3')* PmeI-BGHpolyA-Fw Gtttaaacgcctcgactgtgccttctagttg (4) SpeI-BGHpolyA-Rv Actagttccccagcatgcctgctatt (5) BamHI/KpnI-EGFP-Fw Aatggatccggtaccaccatggtgagcaagggcg (6) NruI-EGFPstop-Rv Ggcacttcgcgattacttgtacagctcgtccatgcc (7) NruI-RMD-Fw Ttatctcgcgaaccatgactcaacgcttgttcg (8) RMD-BsuDel-Rv Atcacggaatgcctcgggtacgtatg (9) RMD-BsuDel-Fw Gctggtcaaacatacgtacccgaggc (10) ApaI-RMD-Rv Tgggcccttactcctctctaacacgagattccca (11) EcoRI-BGHpolyA-Fw Attgtgaattcgcctcgactgtgccttctagttgc (12) EcoRV-BGHpolyA-Rv Taattgatatctccccagcatgcctgctattg (13) NheI-ppol-Fw Gccgcgctagcatcatggagataattaaaatgataaccatctcgcaaataa (14) NotI-ppol-Rv Aatttgcggccgcagcgcccgatggtgggacg (15) XhoI-ppol-Fw Gggcctcgagatcatggagataattaaaatgataaccatctcgcaaataa (16) SphI/PmeI-ppol-Rv Atttgcatgcgtttaaacagcgcccgatggtgggacg (17) NheI-p6.9-Fw Gccgcgctagcaaattccgttttgcgacgatg (18) NotI-p6.9-Rv Aatttgcggccgcgtttaaattgtgtaatttatgtagctgtaatttttacc (19) XhoI-p6.9-Fw Gggcctcgagaaattccgttttgcgacgatg (20) SphI/PmeI-p6.9-Rv Atttgcatgcgtttaaacgtttaaattgtgtaatttatgtagctgtaatttttacc (21) orfl24-Fw Atttgtatttaatcaatcgaaccgtgcac (22) RMD-Rv Gattgggaatctcgtgttagagaggagtaa (23) EGFP-Fw Atcttcttcaaggacgacggcaac (24) gp64-Rv Agcaagatggtaagcgctattgttttatatgtgc (25) chi-Fw Agatgggtatgaaaccatacaacaagtgtg (26) cath-Rv Cgctaccataatctttgttgaatcgatg (27) *numbers in parentheses are SEQ ID NOS:

Cells and Viruses

[0053] Sf9 and Tni PRO.TM. (Expression Systems, Woodland, Calif.) cells were routinely maintained as shake-flask cultures in ESF 921 medium (Expression Systems) at 28.degree. C. High Five.TM. cells (Life Technologies) were maintained as adherent cultures in TNM-FH medium containing 10% (v/v) fetal bovine serum (Atlanta Biologics, Atlanta, Ga.).

[0054] SfRMD is a transgenic Sf9 cell derivative that was produced for this study using a modification of an established procedure (Harrison and Jarvis, 2007). Briefly, Sf9 cells were co-transfected with a mixture of pIE1-Neo (Jarvis et al. 1990) and pIE1-RMD using a modified calcium phosphate method (Summers and Smith 1987). The transfected cells were allowed to recover for 1 day, treated with 1 mg of G418 (Life Technologies)/mL of TNM-FH medium containing 10% (v/v) fetal bovine serum (Atlanta Biologics) for 1 week, and G418-resistant clones were isolated by limiting dilution, as described previously (Hollister and Jarvis 2001). After stepwise amplification into larger cultures, individual clones were assayed by cell surface staining with a fucose-specific lectin, as described below. Unstained clones were designated SfRMD, characterized in more detail, and used for downstream experiments, as described below.

[0055] TnRMD is a transgenic High Five.TM. cell derivative that was designed to constitutively express the Rmd gene and produced for this study as described above for SfRMD. The baculovirus expression vector designated AcmIgG2a-Fc has been described previously (Geisler and Jarvis 2012).

[0056] The baculovirus expression vector designated AcP(+)IE1-RMD was isolated by co-transfecting Sf9 cells with a mixture of pAcP(+)IE1-RMD and BacPAK6 (Kitts and Possee 1993) baculoviral DNA after pre-linearizing the latter by digestion with Bsu36I. Viral progeny were harvested, clones were resolved by plaque assay in the presence of X-Gal (Sigma-Aldrich; St. Louis, Mo.), and an isolated clone with a white plaque phenotype was amplified and titered in Sf9 cells.

[0057] Baculovirus expression vectors designated Ac-.alpha.CD20-IgG and Acp6.9-.alpha.CD20-IgG were isolated by co-transfecting Sf9 cells with mixtures of either pVL1393-polh-.alpha.CD20-IgG or pVL1393-p6.9-.alpha.CD20-IgG and AcGT baculoviral DNA (Toth et al. 2011) after pre-linearizing the latter by digestion with Bsu36I. The transfected cells were cultured for 5 days in growth medium containing 100 .mu.M ganciclovir (Life Technologies) and then viral progeny were harvested, clones were resolved by plaque assay, and an isolated clone with a white plaque phenotype was amplified and titered in Sf9 cells, as described above.

[0058] The baculovirus expression vector designated AcRMD was isolated by co-transfecting Sf9 cells with a mixture of p.DELTA.Chi/Cath-EGFP/RMD.DELTA.Bsu36I and BacPAK6 (Kitts and Possee 1993) baculoviral DNA (FIG. 14). This recombination strategy was custom-designed to replace the AcMNPV chiA and v-cath coding sequences with Rmd and EGFP coding sequences placed under the control of dual AcMNPV ie1 promoters separated by the AcMNPV hr5 enhancer. Viral progeny were harvested, clones were resolved by plaque assay, and fluorescent plaques were identified using an Axiovert 25 microscope equipped with HBO 50 epi-fluorescence unit (Carl Zeiss, Oberkochen, Germany). Several plaques were picked, screened by PCR, as described below, and those that included viral progeny with the Rmd and EGFP genes in the correct genomic location were used for a second round of plaque purification. The PCR screen was repeated, promising clones were used for a third round of plaque purification, and well-isolated clones with fluorescent plaque phenotypes were picked and amplified in Sf9 cells. A Wizard genomic DNA purification kit (Promega) was used to extract total DNA from Sf9 cells infected with each of those viral clones and used as the templates for final PCR analyses, which were the same as those performed for the prior screening steps. In each case, we performed PCRs with two different primer pairs, orf124-Fw and RMD-Rv or EGFP-Fw and gp64-Rv to determine if the final AcRMD isolates had the EGFP/RMD.DELTA.Bsu36I expression cassette in the correct genomic location, that is, in place of the viral chiA and v-cath genes. In addition, we used a third primer pair, chi-Fw and cath-Rv, to directly confirm the absence of those viral genes and show that the final AcRMD isolates were not detectably contaminated with parental BacPAK6 vector DNA. Three clones were positive with the first two primer pairs and negative with the third and one clone was designated AcRMD, further amplified in Sf9 cells, and used for the remainder of this study.

[0059] Baculovirus expression vectors designated AcRMD-.alpha.CD20-IgG and AcRMDp6.9-.alpha.CD20-IgG (FIGS. 15 and 16) were isolated by co-transfecting Sf9 cells with a mixture of pVL1393-polh-.alpha.CD20-IgG or pVL1393-p6.9-.alpha.CD20-IgG and AcRMD baculoviral DNA pre-linearized by digestion with Bsu36I. Viral progeny were harvested, clones were resolved by plaque assay, and an isolated clone with a white plaque phenotype was amplified and titered in Sf9 cells, as described above.

Cell Surface Lectin Staining

[0060] Sf9, SfRMD, High Five.TM., or TnRMD cells were stained with a mixture of biotinylated AAL (Vector Labs, Burlingame, Calif.) and fluorescein-conjugated streptavidin (Vector Labs) in lectin staining buffer (10 mM Hepes, pH 7.5, 50 mM NaCl, mM CaCal.sub.2, 1 mM MgCl.sub.2, 1 mM MnCl.sub.2) for 10 min at room temperature. The cells were then washed twice with lectin staining buffer and imaged using an Olympus FSX 100 fluorescence microscope (Tokyo, Japan).

Recombinant Protein Expression and Purification

[0061] For small scale recombinant protein expression experiments, Sf9 cells were seeded at a density of 2.times.10.sup.6 cells/well into 6-well plates and infected with Ac-.alpha.CD20-IgG, Acp6.9-.alpha.CD20-IgG, AcRMD-.alpha.CD20-IgG, or AcRMDp6.9-.alpha.CD20-IgG at a multiplicity of about 2 plaque forming units/cell. The virus was allowed to adsorb for 1 hour and then the infected cells were washed once with 1 mL of ESF 921 and cultured in 1 mL of fresh ESF 921 at 28.degree. C. to 48 hours post-infection. At that time, the cell-free media were prepared by low speed centrifugation and used as the extracellular fractions for downstream analysis. The cell pellets were washed once with phosphate buffered saline, lysed with 1 mL of extraction buffer (20 mM HEPES, pH 7.4, 0.15 M NaCl, 0.1 mM EDTA, 0.5% Nonidet P-40), clarified by centrifugation at 13,000 rpm for 10 min, and the supernatants were used as the intracellular fractions for downstream analysis.

[0062] For larger scale recombinant protein expression and purification, Sf9, Tni PRO.TM., or High Five.TM. cells were seeded into 50 mL shake flask cultures at a density of 2.times.10.sup.6 cells/mL in ESF 921 and then infected with Acp6.9-.alpha.CD20-IgG, AcRMDp6.9-.alpha.CD20-IgG, AcmIgG2a-Fc, or a mixture of AcmIgG2a-Fc and AcP(+)IE1-RMD using a multiplicity of about 2 plaque forming units/cell for each virus. After a 1 hour adsorption period, the infected cells were gently pelleted, resuspended in 50 mL of fresh ESF 921 supplemented with antibiotics (1.25 .mu.g/mL amphotericin B and 25 .mu.g/mL gentamicin), returned to the shake flasks, and incubated at 28.degree. C. to 48 hours post-infection. Cells and debris were pelleted by centrifugation at 1,000.times.g for 10 min at 4.degree. C., the supernatants were harvested, and budded baculovirus progeny were removed by centrifugation at 70,000.times.g for 30 min at 4.degree. C. One Complete Protease Inhibitor Cocktail tablet (Roche Diagnostics, Indianapolis, Ind.) was dissolved in each final supernatant and then each was transferred into a 12-14,000 molecular weight cut-off membrane (Spectrum Labs, Rancho-Dominguez, Calif.) and dialyzed against 0.1 M NaCl for 6 hours. Each anti-CD20-IgG or mIgG2a-Fc preparation was subsequently dialyzed against 0.15 M or 0.5 M NaCl, respectively, and then each was purified by was affinity-purified Protein A agarose (GenScript, Piscataway, N.J.) or ProBond nickel (Life Technologies) affinity chromatography, respectively, according to the manufacturer's instructions. Eluted fractions containing the purified proteins were pooled and desalted on PD10 columns (GE Healthcare) equilibrated with phosphate buffered saline.

SDS-PAGE, Western Blotting, and Lectin Blotting Analyses

[0063] Several different types of samples were isolated from SD, Tni PRO.TM., and/or High Five.TM. cells and used for SDS-PAGE, western blotting, and/or lectin blotting analysis. These included intracellular fractions from uninfected cells, extracellular fractions from cells infected with various recombinant baculoviruses, and anti-CD20-IgG or mIgG2a-Fc purified from cells infected with various recombinant baculoviruses, as described above. In some cases, the samples were pre-treated with PNGase-F in reaction buffer (New England Biolabs) or PNGase-F reaction buffer alone according to the manufacturer's instructions, as described in the Figure legends. All samples were boiled in Laemmli sample buffer and then proteins were resolved by SDS-PAGE on 12% polyacrylamide gels and either stained with Coomassie Brilliant blue, destained, and imaged or electrophoretically transferred to Immobilon-P membranes (Millipore). The latter were blocked for 1 hour at room temperature with Tris-buffered saline (150 mM NaCl in 50 mM Tris-HCl, pH 7.5) containing either 5% bovine serum albumin (w/v; Sigma-Aldrich) and 0.5% (v/v) Tween 20 (Sigma-Aldrich) or 1% Tween 20 for western or lectin blotting assays, respectively. After blocking, western blotting assays were completed using alkaline phosphatase-conjugated goat anti-human IgG .kappa. chain (Sigma-Aldrich) to detect the anti-CD20-IgG light chain, alkaline phosphatase-conjugated goat anti-human IgG Fc (Sigma-Aldrich) to detect the anti-CD20-IgG heavy chain, alkaline phosphatase-conjugated goat anti-mouse IgG to detect mIgG2a-Fc, or rabbit anti-HRP IgG (Gentaur, Brussels, Belgium) as the primary and alkaline phosphatase-conjugated goat anti-rabbit IgG (Sigma-Aldrich) as the secondary antibody to detect core .alpha.1,3-liked fucose. The lectin blotting assays were completed using alkaline phosphatase-conjugated AAL (Vector Laboratories) and the probes used for all western and lectin blotting assays were detected using a standard chromogenic assay for alkaline phosphatase activity (Blake et al. 1984).

Mass Spectrometry

[0064] N-glycans were enzymatically released from various purified mIgG2a-Fc and anti-CD20-IgG preparations by exhaustive digestion with PNGaseAr (New England Biolabs). The spent reactions were applied to pre-conditioned C18 SepPak cartridges (Waters Corp., Milford, Mass.) and the flow-through and a 5% (v/v) aqueous acetic acid wash were pooled, evaporated, and permethylated, as described previously (Dell et al. 1994). The permethylated N-glycan derivatives were extracted into chloroform, pooled with several aqueous washes, re-evaporated, and then resuspended in acetonitrile, mixed 1:1 with 2,5-dihydroxybenzoic acid matrix (10 mg/mL in 50% aqueous acetonitrile), and samples were spotted onto the MALDI-TOF target plate. Data acquisition was performed manually on a Model 4700 Proteomics Analyzer equipped with an Nd:YAG laser (Applied Biosystems, Framingham, Mass.) and 1,000 shots were accumulated in the reflectron positive ion mode.

[0065] The following example is provided to illustrate certain embodiments of the invention. It is not intended to limit the invention in any way.

EXAMPLE I

A Novel Baculovirus Vector for the Production of Non-Fucosylated Recombinant Glycoproteins in Insect Cells

[0066] Analysis of Core .alpha.1,3-Fucosylation in Three Insect Cell Lines

[0067] High Five.TM. cells, derived from Trichoplusia ni, but not Sf9 cells, derived from Spodoptera frugiperda, produce core .alpha.1,3-fucosylated glycoproteins (Rudd et al. 2000; Seismann et al. 2010; Blank et al. 2011; Palmberger et al. 2011). Another Trichoplusia ni cell line used as a host for baculovirus expression vectors is Tni PRO.TM. (Kwon et al. 2009; Bourhis et al. 2010; Bongiovanni et al. 2012; He et al. 2013; Merchant et al. 2013), but its capacity for core .alpha.1,3-fucosylation has not been reported. Thus, we analyzed intracellular extracts of uninfected Tni PRO.TM. cells by western blotting with anti-horseradish peroxidase (HRP), which detects core .alpha.1,3-linked fucosylation, using extracts from Sf9 and High Five.TM. cells as negative and positive controls. Coomassie brilliant blue staining showed that approximately equal amounts of protein were loaded in each case (FIG. 2A). The anti-HRP antibody did not detectably react with the Sf9 lysates, but reacted with several glycoproteins in the High Five.TM. lysates, as expected (FIG. 2B). In addition, this antibody reacted with several glycoproteins in the Tni PRO.TM. lysates (FIG. 2B), indicating that Tni PRO.TM. cells produce the immunogenic core .alpha.1,3-fucosylated sugar epitope at levels roughly comparable to High Five.TM. cells. These results show that it will be necessary to block core .alpha.1,3-fucosylation in both of these cell lines before we can exploit their potentially higher capacity for recombinant glycoprotein production (Davis et al. 1992; Krammer et al. 2010).

[0068] Glycoengineering Insect Cells to Block Glycoprotein Fucosylation

[0069] Our plan to block glycoprotein fucosylation in insect cell lines focused on blocking the biosynthesis of GDP-L-fucose, which is the donor substrate required for this process. This was a particularly attractive approach in our system because insects appeared to be the only multicellular organisms lacking two enzymes, FUK and FPGT, required for the GDP-L-fucose salvage pathway in other organisms (FIG. 1B). We drew this conclusion from a previous study indicating there are no FUK and FPGT orthologs in the Drosophila melanogaster genome, which was the only insect genome sequenced at that time (Rhomberg et al. 2006). However, because we now have more information from silkworm, honeybee, and mosquito genome sequencing projects, among others, we also searched the NCBI database using mammalian FUK and/or FPGT genes as queries. We identified putative orthologs in some invertebrates, including arthropods and nematodes, but none in any insects (FIG. 3A-3B). In contrast, using genes required for de novo GDP-L-fucose synthesis as queries, we found putative orthologs in a wide variety of insects, as expected (FIG. 3C-3D). Although we could not exclude the possibility that insects have an unknown salvage pathway, these results strengthened the idea that we could effectively block GDP-L-fucose biosynthesis by blocking the de novo biosynthetic pathway, alone, in insect cell lines.

[0070] In principle, we might have achieved this goal by inactivating any of the genes encoding enzymes involved in this pathway, including GMD, Fx, GFR, or FUT8 (FIG. 1B). However, there are no reported examples of targeted gene knockouts in any lepidopteran insect cell line and this approach is technically complicated by the fact that neither the Spodoptera frugiperda nor the Trichoplusia ni genomes have been sequenced. On the other hand, we have reported many examples of foreign gene knock-ins using both Sf9 (Hollister et al. 1998; Hollister and Jarvis 2001; Hollister et al. 2002; Aumiller et al. 2003; Aumiller et al. 2012; Geisler and Jarvis 2012; Mabashi-Asazuma et al. 2013) and High Five.TM. (Breitbach and Jarvis 2001) cells, as part of our broader effort to glycoengineer the baculovirus-insect cell system. Thus, we pursued an analogous glycoengineering strategy that involved transforming Sf9 and High Five.TM. cells with a constitutively expressible Pseudomonas aeruginosa Rmd gene. This gene encodes an enzyme that consumes the immediate precursor to GDP-L-fucose to produce GDP-D-rhamnose, which we believed would be a dead-end product in insect cells (FIG. 1B; Rocchetta et al. 1998). Thus, we expected Rind to block the production of GDP-L-fucose and glycoprotein fucosylation because GDP-L-fucose is required as the donor substrate for this process.

[0071] We constructed an expression plasmid encoding Rmd under the transcriptional control of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) immediate early 1 (ie1) promoter, as described in Materials and methods. We then co-transfected SD and High Five.TM. cells with a mixture of this plasmid and an antibiotic-resistance marker and selected polyclonal transformed Sf9 and High Five.TM. cell subpopulations, which were designated SfRMD and TnRMD, respectively. We assayed both polyclonal transformed cell populations for cell surface fucosylation by staining with Aleuria aurantia lectin (AAL), as described in Materials and methods. The results showed that 100% of the parental Sf9 and High Five.TM. cells were stained, while only 23% of the SfRMD and 33% of the TnRMD cells were stained with AAL (FIG. 4). These results demonstrated that Rmd overexpression significantly reduced glycoprotein fucosylation in insect cells. We subsequently isolated single cell clones from the polyclonal transformed SfRMD and TnRMD populations, identified several that exhibited no detectable AAL cell surface staining, and amplified one of each, designated SfRMD 2B2 and TnRMD 6A6. We then infected Sf9, SfRMD 2B2, High Five.TM. and TnRMD 6A6 cells with a baculovirus vector encoding a 6X HIS-tagged Fc domain of mouse IgG2a (mIgG2a-Fc) under the control of the baculovirus p6.9 promoter, which is activated during the late phase of infection (Passarelli and Guarino 2007), and affinity-purifed the mIgG2a-Fc from the extracellular fractions, as described in Materials and methods. Samples were then treated with peptide-N.sup.4-(N-acetyl-.beta.-glucosaminyl)asparagine amidase (PNGase)-F or reaction buffer alone, resolved by SDS-PAGE, transferred to a PVDF membrane, and probed with AAL to detect fucose or with anti-mouse IgG to detect the protein (FIG. 5).

[0072] The results of this analysis showed that the mIgG2a-Fc preparations produced by the parental cell lines were AAL-reactive and either completely (Sf9) or partially (High Five.TM.) sensitive to PNGase-F, indicating they were core .alpha.1,6-(Sf9) or .alpha.1,6- and .alpha.1,3-(High Five.TM.) fucosylated, respectively. In contrast, neither of the mIgG2aFc preparations from SfRMD 2B2 or TnRMD 6A6 cells was AAL-reactive, indicating that RMD effectively blocked glycoprotein fucosylation in both cell types. However, we were surprised to find that both of these prototype SfRMD and TnRMD clones recovered strong AAL reactivity after 38 and 35 passages in culture, respectively, indicating that the fucosylation-negative phenotype is unstable in these cells (data not shown). We obtained this same result with other SfRMD and TnRMD clones, which was extremely surprising, because this knock-in approach had been used previously to isolate a stable, fucosylation deficient CHO cell derivative (von Horsten et al. 2010) and because we had previously demonstrated that a transgenic insect cell line transformed with six different transgenes was stable for at least 300 passages in culture (Aumiller et al. 2012). Although we found that they still expressed the Rmd gene at the transcriptional level, we did not expend any further effort to determine how the SfRMD and TnRMD cells recovered their original fucosylation-positive phenotypes. Rather, after finding, much to our surprise, that our insect cell glycoengineering approach was unsuccessful, we began to develop a new approach for blocking glycoprotein fucosylation in the baculovirus-insect cell system.

[0073] Assessing a Vector-Based Glycoengineering Strategy for Blocking Glycoprotein Fucosylation

[0074] In view of the surprising phenotypic instability of SfRMD and TnRMD cells, we abandoned our efforts to glycoengineer the host cell component and focused our attention on the baculoviral vector component of the baculovirus-insect cell system. The baculovirus-insect cell system is a transient expression system in which baculovirus-infected insect cells express the glycoprotein of interest for a period of about 2 days, beginning about a day after infection, when the gene encoding the glycoprotein of interest begins to be expressed during the late or very late phase of infection. Baculovirus-infected cells are typically harvested by about 60-72 hours post-infection because the host cells die and lyse at later times of infection. Given the transient nature of this expression system, we realized that the phenotypic instability observed with long-term constitutive expression of Rmd in the host cells could be overcome by engineering the viral vector to express this enzyme. However, we also realized we would have to design the new vector to express Rmd early in infection, in order to reduce endogenous GDP-L-fucose to low enough levels to block fucosylation before the glycoprotein of interest began to be expressed during a later phase of infection.

[0075] To assess our ability to meet these requirements, we isolated a recombinant baculovirus designated AcP(+)IE1-RMD, which encodes Rmd under the control of the AcMNPV ie1 promoter and was expected to express the Rmd gene immediately after baculovirus infection (Guarino and Summers 1986). We then used this virus to examine the impact of immediate early Rmd expression on fucosylation of mIgG2a-Fc. This was accomplished by co-infecting Sf9 or Tni PRO.TM. cells with equal doses of AcP(+)IE1-RMD and Acp6.9-mIgG2a-Fc, or with Acp6.9-mIgG2a-Fc alone as a control, and then affinity-purifying the mIgG2a-Fc from the extracellular fractions for analysis, as described in Materials and methods. Samples of the purified mIgG2a-Fc were then treated with PNGase-F or reaction buffer alone, resolved by SDS-PAGE, transferred to a PVDF membrane, and probed with AAL to detect fucose, anti-HRP to detect core .alpha.1,3-linked fucose, or anti-mouse IgG to detect the protein. The results showed that the control mIgG2a-Fc preparation produced by Sf9 cells infected with Acp6.9-mIgG2a-Fc alone were AAL-reactive (FIG. 6, top panel), indicating it was fucosylated. In addition, PNGase-F pre-treatment eliminated its AAL reactivity (FIG. 6, top panel), indicating that it was exclusively core .alpha.1,6-fucosylated, because PNGase-F does not remove core .alpha.1,3-fucosylated N-glycans (Tretter et al. 1991). This interpretation was supported by the fact that anti-HRP, which recognizes the immunogenic sugar epitope produced by core .alpha.1,3-fucosylation, did not react with this mIgG2a-Fc preparation (FIG. 6, middle panel). In contrast, the mIgG2a-Fc preparation from Sf9 cells co-infected with Acp6.9-mIgG2a-Fc and AcP(+)IE1-RMD was not AAL-reactive (FIG. 6, upper panel), indicating it was not fucosylated and, therefore, that baculovirus-mediated Rmd expression blocked core .alpha.1,6-fucosylation in Sf9 cells. The mIgG2a-Fc Fc from Tni PRO.TM. cells infected with Acp6.9-mIgG2a-Fc alone reacted not only with AAL (FIG. 6, top panel), but also with anti-HRP (FIG. 6, middle panel), whether or not it was pre-treated with PNGase-F, indicating that it was core .alpha.1,3-fucosylated. This interpretation was supported by the fact that PNGase-F did not detectably alter the electrophoretic mobility of this mIgG2a-Fc preparation (FIG. 6, bottom panel). In contrast, the mIgG2a-Fc from Tni PRO.TM. cells co-infected with Acp6.9-mIgG2a-Fc and AcP(+)IE1-RMD had no detectable AAL (FIG. 6, top panel) or anti-HRP (FIG. 6, middle panel) reactivity and was sensitive to PNGase-F (FIG. 6, bottom panel). These results indicated that baculovirus-mediated Rmd expression blocked both core .alpha.1,6- and core .alpha.1,3-fucosylation in Tni PRO.TM. cells.

[0076] To more directly assess the impact of AcP(+)IE1-RMD co-infection on core fucosylation of mIgG2a-Fc, we enzymatically released, permethylated, and analyzed the N-glycans from each mIgG2a-Fc preparation in FIG. 6 by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), as described in Materials and methods. The results indicated that the major N-glycans isolated from the mIgG2a-Fc produced by Sf9 cells infected with Acp6.9-mIgG2a-Fc alone were mono-fucosylated (m/z 1345.7 and 1590.8; FIG. 7A). Based on the results shown in FIG. 6, these are most likely core .alpha.1,6-fucosylated N-glycans. In contrast, none of the peaks assigned as fucosylated N-glycans were detected in the mIgG2a-Fc produced by Sf9 cells co-infected with Acp6.9-mIgG2a-Fc and AcP(+)IE1-RMD (FIG. 7B). The major N-glycans isolated from the mIgG2a-Fc produced by Tni PRO.TM. cells infected with Acp6.9-mIgG2a-Fc alone included mono-, but predominantly di-fucosylated structures (FIG. 7C). While their masses cannot directly reveal the nature of the linkages between the fucose and core N-acetylglucosamine residues in these N-glycans, it is most reasonable to conclude that the di-fucosylated N-glycans have both .alpha.1,6- and .alpha.1,3-linked fucose residues. Again, none of the peaks assigned as mono- or di-fucosylated N-glycans were detected in the mIgG2a-Fc produced by Tni PRO.TM. cells co-infected with Acp6.9-mIgG2a-Fc and AcP(+)IE1-RMD (FIG. 7D). Together, the AAL blotting, anti-HRP antibody blotting, and MALDI-TOF MS data clearly demonstrate that baculovirus-mediated expression of Rmd at an early stage of infection can effectively block core .alpha.1,6- and .alpha.1,3-fucosylation of recombinant glycoproteins expressed later in infection in the baculovirus-insect cell system.

A Novel Baculovirus Vector Designed to Produce Non-Fucosylated Recombinant Glycoproteins

[0077] While co-infection with multiple baculoviruses is an approach that can be used to co-express two or more different recombinant proteins in insect cells, balanced co-infections can be difficult to achieve, infection with multiple baculoviruses can dramatically reduce recombinant glycoprotein yields, and optimal co-infection conditions must be established for each experiment (Sokolenko et al. 2012). We chose to circumvent these issues by isolating a novel baculovirus vector designed not only to express Rmd immediately after infection, but also to facilitate downstream insertion of genes encoding any glycoprotein of interest for expression at a later time of infection. The experimental design used to isolate this new baculovirus vector, designated AcRMD, included using the ie1-Rmd gene to replace portions of the viral chiA and v-cath genes, which encode a chitinase purported to interfere with secretory pathway function (Hawtin et al. 1997; Kaba et al. 2004; Hitchman et al. 2010) and a cathepsin-like protease that can degrade recombinant proteins of interest (Slack et al. 1995; Kaba et al. 2004; Hitchman et al. 2010), respectively. The complete details of the strategy used to isolate this new vector are given in Materials and methods and genetic maps of the parental viral genome, transfer plasmid, and final baculoviral vector are shown in FIG. 8 and the plasmid map shown in FIG. 14.

[0078] AcRMD Produces Non-Fucosylated Recombinant Glycoproteins

[0079] To examine the capabilities of the new baculovirus vector described in the preceding section, we isolated two independent daughter recombinants encoding rituximab, an anti-CD20-IgG, with the light and heavy chain genes placed under the independent transcriptional control of dual back-to-back promoters from either the AcMNPV p6.9 or polyhedrin genes (see genetic maps in FIG. 9). We chose rituximab as the model glycoprotein for this work because it is a biotechnologically relevant therapeutic antibody that is core .alpha.1,6-fucosylated in mammalian cells, has enhanced effector function in the absence of fucosylation, and has been used to treat rheumatoid arthritis and non-Hodgkin's lymphoma in human patients (Li et al. 2013; reviewed by Lim et al. 2010). We isolated AcRMD daughters encoding anti-CD20-IgG under the control of either the p6.9 or the polyhedrin promoters because the latter is activated during the very late phase of infection and is widely used to drive recombinant gene expression in baculoviral vectors, whereas the former is activated somewhat earlier, during the late phase of infection, and can sometimes provide higher efficiencies of protein secretion (Sridhar et al. 1993; Rankl et al. 1994; Bozon et al. 1995; Kost et al. 1997; Toth et al. 2011). We then infected Sf9 cells with these viruses, designated Ac-.alpha.CD20-IgG, Acp6.9-.alpha.CD20-IgG, AcRMD-.alpha.CD20-IgG, or AcRMDp6.9-.alpha.CD20-IgG, and measured the relative amounts of anti-CD20-IgG produced and secreted by viruses encoding the heavy and light chains under the control of the two different promoters. The results showed that all four baculovirus vectors induced production and secretion of the anti-CD20-IgG heavy and light chains (FIG. 10). The results also showed that cells infected with the baculovirus vectors encoding anti-CD20-IgG under the control of the late p6.9 promoters secreted higher levels of heavy and light chains than those infected with the baculovirus encoding this product under the control of the very late polyhedrin promoters. Together with the lower levels of heavy and light chains observed in the intracellular fraction, these results indicated that the p6.9-based vectors provided higher efficiencies of anti-CD20-IgG secretion than the polyhedrin-based vectors (FIG. 10). Based on these results, we used Acp6.9-.alpha.CD20-IgG and AcRMDp6.9-.alpha.CD20-IgG for the remainder of the experiments described in this study.

[0080] Acp6.9-.alpha.CD20-IgG or AcRMDp6.9-.alpha.CD20-IgG were used to infect Sf9 or Tni PRO.TM. cells and the anti-CD20-IgG secreted by all four virus-cell combinations was affinity purified, resolved by SDS-PAGE under reducing or non-reducing conditions with commercial human IgG as a control, and then stained with Coomassie Brilliant Blue. The results showed that each anti-CD20-IgG preparation analyzed under reducing conditions contained approximately equal proportions of the heavy and light chains, indicating that each virus-cell combination produced and secreted properly assembled forms of anti-CD20-IgG (FIG. 11). This was confirmed by SDS-PAGE analysis under non-reducing conditions, which revealed that each anti-CD20-IgG preparation migrated with relative electrophoretic mobilities that were consistent with their calculated molecular weights (.about.165 kDa) and comparable to the human IgG control (FIG. 11). Further analysis showed that the anti-CD20-IgG heavy chain produced by Acp6.9-.alpha.CD20-IgG-infected Sf9 cells was PNGase-F-sensitive and AAL-reactive, indicating that it had core .alpha.1,6-fucosylated N-glycans (FIG. 12). In contrast, the anti-CD20-IgG heavy chain produced by AcRMDp6.9-.alpha.CD20-IgG-infected Sf9 cells was PNGase-F-sensitive, but not AAL-reactive, indicating that it had non-fucosylated N-glycans (FIG. 12). The anti-CD20-IgG heavy chain produced by Acp6.9-.alpha.CD20-IgG-infected Tni PRO.TM. cells was PNGase-F-resistant and AAL-reactive, presumably because its N-glycans were .alpha.1,3-fucosylated in these cells (FIG. 12). In contrast, the anti-CD20-IgG heavy chain produced by AcRMDp6.9-.alpha.CD20-IgG-infected Tni PRO.TM. cells was PNGase-F-sensitive and did not react with AAL (FIG. 12). Together, these results strongly suggested that early expression of Rmd by the vectors derived from the AcRMD parent blocked core fucosylation of anti-CD20-IgG in the baculovirus-insect cell system.

[0081] To confirm and extend these results using a more direct approach, we enzymatically released, permethylated, and used MALDI-TOF MS to analyze the N-glycans from anti-CD20-IgG preparations purified from various virus-cell combinations, as described in Materials and methods. The N-glycan profiles observed with the anti-CD20-IgG from Acp6.9-.alpha.CD20-IgG-infected Sf9 cells included four peaks assigned as mono-fucosylated N-glycans (m/z 1345.7, 1590.8, 1794.9, and 1999.0; FIG. 13A), which are presumably core .alpha.1,6-fucosylated, based on the PNGase-F-sensitivity of the heavy chain (FIG. 12). In contrast, none of these mono-fucosylated peaks were detected among the N-glycans isolated from the anti-CD20-IgG produced by AcRMDp6.9-.alpha.CD20-IgG-infected Sf9 cells (FIG. 13B). In addition, the loss of the fucosylated N-glycan peaks was accompanied by increased N-glycan peaks corresponding to their non-fucosylated counterparts, especially those with m/z values of 1171.6 and 1416.7 (FIGS. 13A and 13B). The N-glycan profiles observed with the anti-CD20-IgG from Acp6.9-.alpha.CD20-IgG-infected Tni PRO.TM. cells included seven peaks assigned as fucosylated N-glycans, three of which were mono-fucosylated (m/z 1345.7, 1590.8, and 1835.9) and four of which were di-fucosylated (m/z 1315.7, 1519.8, 1764.9, and 2173.1; FIG. 13C). The di-fucosylated N-glycans represented .about.65.5% of total (FIG. 13C), reflecting the high levels of core .alpha.1,3-fucosylation observed in Tni PRO.TM. and High Five.TM. cells (FIG. 2). In contrast, no fucosylated N-glycans were detected in the profiles obtained with the anti-CD20-IgG produced by AcRMDp6.9-.alpha.CD20-IgG-infected Tni PRO.TM. cells (FIG. 13D) and, again, the loss of fucosylated N-glycan peaks was accompanied by increases in the N-glycan peaks corresponding to their non-fucosylated counterparts (FIGS. 13C and 13D; m/z 1171.6 and 1416.7). It is tempting to speculate that the large increase in the m/z 1416.7 peak, which represents an N-glycan with a single N-acetylglucosamine residue on its non-reducing end, reflects the relative inability of Sf-FDL (Geisler et al., 2008) to remove this sugar from non-fucosylated substrates. Regardless, the mass spectrometric analysis of the N-glycans isolated from anti-CD20-IgG produced by the Rmd-negative or Rmd-positive baculovirus vectors clearly demonstrated that the new vector designed to express Rmd early in infection can block recombinant glycoprotein fucosylation in the baculovirus-insect cell system.

DISCUSSION

[0082] Core .alpha.1,3-fucosylation generates an immunogenic sugar epitope that has significantly hindered development and utilization of insect-based systems, including the baculovirus-insect cell system for the production of recombinant glycoproteins for therapeutic drug and diagnostic applications in human medicine. In addition, core .alpha.1,6-fucosylation of certain types of recombinant antibodies in this system and others represses their effector functions. Thus, the basic purpose of this study was to develop new tools that could be used to produce non-fucosylated recombinant glycoproteins, including antibodies, in insect-based systems, including the baculovirus-insect cell system.

[0083] It is well established that High Five.TM., which is a widely used insect cell line derived from Trichoplusia ni, produces high levels of immunogenic core .alpha.1,3-fucosylated N-glycans. In the first part of this study, we showed that Tni PRO.TM. cells, also derived from Trichoplusia ni, produce high levels of immunogenic core .alpha.1,3-fucosylated N-glycans, as well. This finding is relevant because High Five.TM. and Tni PRO.TM. cells can potentially produce recombinant glycoproteins at higher levels than other insect cell lines (Davis et al. 1992; Krammer et al. 2010). Tni PRO.TM. cells have the additional advantage of being easier to culture in suspension and, unlike High Five.TM. cells (Dee et al. 1997; Taticek et al. 1997; Savary et al. 1999), are directly transferrable without adaptation from serum containing to serum-free ESF 921 medium (unpublished observations). In the course of this study, we found that a mouse IgG2a-Fc domain and a therapeutic anti-CD20-IgG were both core .alpha.1,6-fucosylated when produced in Sf9 cells and core .alpha.1,6- and core .alpha.1,3-fucosylated when produced in High Five.TM. or Tni PRO.TM. cells. While these results were not surprising in view of previous literature, they were important because they clearly justified an effort to block recombinant glycoprotein fucosylation in the baculovirus-insect cell system.

[0084] To accomplish this goal, we focused on a bacterial enzyme, Rmd, which consumes the direct precursor to GDP-L-fucose and was expected to block recombinant glycoprotein fucosylation in insect cell lines. In fact, previous work had shown that core .alpha.1,6-fucosylation could be blocked in CHO cells genetically transformed to overexpress this enzyme (von Horsten et al. 2010). Thus, our analogous initial approach was to transform Sf9 and High Five.TM. cells to constitutively express Rmd under the control of the AcMNPV ie1 promoter. We successfully isolated Sf9 and High Five.TM. cell subclones that initially had fucosylation-negative phenotypes and were able to produce a non-fucosylated recombinant glycoprotein. However, this phenotype was unstable, as both insect cell lines reverted to fucosylation-positive phenotypes after a relatively small number of passages in culture. This completely surprising result revealed that the cell engineering approach previously used to block core .alpha.1,6-fucosylation in CHO cells cannot be successfully applied to block core .alpha.1,6- and/or .alpha.1,3-fucosylation in insect cell systems. Thus, we sought to develop a new approach that involved glycoengineering the baculovirus vector, rather than the host.

[0085] We assessed the efficacy of our proposed vector engineering approach by co-infecting Sf9 and Tni PRO.TM. cells with separate baculovirus vectors encoding Rmd under the control of the ie1 promoter or mIgG2a-Fc under the control of the polyhedrin promoter. Analysis of the resulting N-glycosylation patterns showed that early expression of Rmd could block core fucosylation of mIgG2a-Fc produced at a later time of infection. This encouraged us to create a novel baculovirus vector designed not only to express Rmd immediately after infection, but also to enable quick and efficient isolation of daughter vectors capable of expressing any recombinant glycoprotein of interest at later times after infection. After isolating and characterizing AcRMD, the parent baculovirus vector, we used it to isolate a daughter encoding the heavy and light chains of rituximab, an anti-CD20-IgG, under the control of dual, back-to-back p6.9 promoters. We chose the late p6.9, rather than the very late polyhedrin promoter to drive expression of this biotechnologically relevant recombinant glycoprotein because we found that it provided a higher efficiency of IgG secretion. Finally, we showed that this novel baculovirus vector could be used to produce recombinant anti-CD20-IgG with no detectable core .alpha.1,6- or .alpha.1,3-fucosylation. This conclusion was based on results obtained from several different methods of N-glycan analysis, including endoglycosidase treatments, lectin blotting assays, and MALDI-TOF MS profiling. It is important to note that our MALDI-TOF MS profiling results indicating there were no detectable fucosylated N-glycans on the recombinant anti-CD20-IgG produced by the Rmd-positive baculovirus vector were obtained using N-glycans isolated with a highly active form of PNGase-A (PNGaseAr; New England Biolabs), which effectively removes core .alpha.1,3-fucosylated structures, and with glycan detection levels in the picomolar range. It is also important to note that our MALDI-TOF MS results revealed no detectable N-glycans containing any deoxyhexose, indicating that GDP-rhamnose is a dead-end product that cannot be utilized for N-glycan modification in the baculovirus-insect cell system.

[0086] There are clear advantages to engineering the baculovirus vector, rather than host cell lines to block recombinant glycoprotein fucosylation in baculovirus-insect cell systems. One is that any investigator familiar with these systems can use the new AcRMD vector in conjunction with an established linearized viral DNA (Kitts and Possee 1993) approach for homologous recombination with familiar, even pre-existing baculovirus transfer plasmids to efficiently isolate daughter baculovirus vectors encoding their favorite recombinant glycoprotein. Another is that the resulting daughter vectors can be used to produce non-fucosylated forms of that product in standard, familiar, commercially available insect cell lines, such as Sf9, Sf21, High Five.TM., Tni PRO.TM., Ea4, S2, or S2R+, even if the investigators favorite cell line normally produces high levels of .alpha.1,3-fucosylated N-glycans. This eliminates the need to maintain specialized cell lines transformed to block recombinant glycoprotein fucosylation, which might require different growth media and/or conditions and have different growth properties, all of which complicate routine cell culture operations. Overall, it is highly advantageous to be able to produce non-fucosylated recombinant glycoproteins by simply replacing the recombinant baculovirus used for standardized production runs with its AcRMD counterpart, without having to alter or re-optimize existing protocols. Most importantly, engineering the virus eliminates the problem of genetic instability associated with engineering the insect cell lines, because low passage virus stocks can be produced, checked, and stored in a biologically inert state. In contrast, cell lines transformed to constitutively express Rmd must be maintained in culture and, therefore, subjected to constant selective pressure, which can drive loss of the fucosylation-negative phenotype, as observed in this study. Mammalian cell expression systems engineered to produce non-fucosylated recombinant glycoproteins, such as FG1, CHO SM 3G1, CHO FUT8.sup.-/-, RMD-CHO, and CHO-DUKX (Imai-Nishiya et al. 2007; Kanda et al. 2007; Malphettes et al. 2010; von Horsten et al. 2010; Zhong et al. 2012), are also subject to the potential problem of long-term instability. In addition, mammalian cells have a salvage pathway that can produce GDP-L-fucose using exogenous fucose, which is a common contaminant of many cell culture components. This salvage pathway can rescue recombinant glycoprotein fucosylation in all mammalian cell lines engineered to eliminate this modification except the FUT8 knockout line. This is unlikely to be a problem in the baculovirus-insect cell system because insects do not appear to encode the enzymes involved in the salvage pathways for GDP-L-fucose biosynthesis.

[0087] Arguably, the most important feature of the novel baculovirus vector described in this study is its ability to eliminate the immunogenic sugar epitope resulting from core .alpha.1,3-fucosylation of recombinant glycoproteins. This will enable investigators to exploit the potentially higher productivity of insect cell lines derived from Trichoplusia ni for recombinant glycoprotein manufacturing (Davis et al. 1992; Krammer et al. 2010). It will also expand the utility of the baculovirus-insect cell system to include production of recombinant glycoproteins for human clinical applications, including therapeutics and diagnostics. Historically, the production of recombinant glycoproteins for therapeutic use in humans has not been a legitimate application of the baculovirus-insect cell system. This study shifts this paradigm because the new baculovirus vector described herein can block core .alpha.1,3-fucosylation in insect cell lines glycoengineered to produce humanized, terminally sialylated N-glycans. The combination of these emerging tools constitutes a novel baculovirus-insect cell platform that can be used to manufacture safe and efficacious glycoproteins for human therapy.

[0088] Another important feature of the novel baculovirus vector described in this study is its ability to block core .alpha.1,6-fucosylation, which is a common modification of a conserved N-glycan on the Fc domain that represses the effector functions of certain types of therapeutic antibodies. As noted above, core .alpha.1,6-fucosylation has been blocked in other expression systems in efforts to produce therapeutic antibodies with enhanced effector functions and, therefore, higher efficacy at lower doses (reviewed by Yamane-Ohnuki and Satoh 2009). Sf9 and Tni PRO.TM. cells infected with the AcRMD daughter vector encoding anti-CD20-IgG produced a non-fucosylated form of this antibody, which is expected to have enhanced effector function, based on a significant body of previous literature (Shields et al. 2002; Shinkawa et al. 2003; Li et al. 2013; reviewed by Lim et al. 2010; Owen and Stewart 2012). Moreover, while many recombinant antibodies have been produced in the baculovirus-insect cell system (reviewed by Cerutti and Golay 2012), most were expressed using dual, back-to-back, very late polyhedrin and p10 promoters to express the heavy and light chains and the heavy chain product. In this study, we found that expression of the heavy and light chains under the control of dual, back-to-back, late p6.9 promoters separated by the second intron from the Drosophila white gene provided a higher secretion efficiency than the analogous arrangement of very late polyhedrin promoters (FIG. 10). This result is consistent with those obtained in similar studies on the relationship between the timing of promoter activation and the efficiency of recombinant glycoprotein processing (Sridhar et al. 1993; Rankl et al. 1994; Bozon et al. 1995; Kost et al. 1997; Toth et al. 2011).

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[0194] While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope of the present invention, as set forth in the following claims.

Sequence CWU 1

1

27111844DNAArtificial SequencepChi/Cath-EGFP/RMD 1ttacacgtag aattctactc gtaaagcagt ttatgagccc gtgtgcaaaa catgacatca 60tctcgatttg aaaaacaaat gaccatcatc cactcgatcg tgcgttacaa gtagaattct 120actcgtaaag ccagttcggt tatgagccgt gtgcaaaaca tgacatcagc ttatgactca 180tacttgattg tgttttacgc gaagggcgaa ttccagcaca ctggcggccg ttactagacg 240atgtctttgt gatgcgcgcg acatttttgt aggttattga taaaatgaac ggatacgttg 300cccgacatta tcattaaatc cttggcgtag aatttgtcgg gtccattgtc cgtgtgcgct 360agcatgcccg taacggacct cgtacttttg gcttcaaagg ttttgcgcac agacaaaatg 420tgccacactt gcagctctgc atgtgtgcgc gttaccacaa atcccaacgg cgcagtgtac 480ttgttgtatg caaataaatc tcgataaagg cgcggcgcgc gaatgcagct gatcacgtac 540gctcctcgtg ttccgttcaa ggacggtgtt atcgacctca gattaatgtt tatcggccga 600ctgttttcgt atccgctcac caaacgcgtt tttgcattaa cattgtatgt cggcggatgt 660tctatatcta atttgaataa ataaacgata accgcgttgg ttttagaggg cataataaaa 720gaaatattgt tatcgtgttc gccattaggg cagtataaat tgacgttcat gttggatatt 780gtttcagttg caagttgaca ctggcggcga caagatcgtg aacaaccaag tgaccggtac 840caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct 900ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac 960ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc 1020caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat 1080gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat 1140cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac 1200cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg 1260gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa 1320gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct 1380cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa 1440ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat 1500ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa 1560gtaatcgaaa cgcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc 1620ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg 1680aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg 1740acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggaacta gtgtggcggc 1800gctaaacaag aaatagcccc ggtggccaag agtatgcccg ttcctcctac ttttaagcta 1860attcgactgg cgtcgtctca acaaagtcac tagcgtaaaa aatcagggca tgtgtggcgc 1920ctgctgggcg tttgccactc tggctagttt ggaaagtcaa tttgcaatca aacataacca 1980gttgattaat ctgtcggagc agcaaatgat cgattgtgat tttgtcgacg ctggctgtaa 2040cggcggcttg ttgcacacag cgttcgaagc catcattaaa atgggcggcg tacagctgga 2100aagcgactat ccatacgaag cagacaataa caattgccgt atgaactcca ataagtttct 2160agttcaagta aaagattgtt atagatacat taccgtgtac gaggaaaaac ttaaagattt 2220gttacgcctt gtcggcccta ttcctatggc catagacgct gccgacattg ttaactataa 2280acagggtatt ataaaatatt gtttcaacag cggtctaaac catgcggttc ttttagtggg 2340ttatggtgtt gaaaacaaca ttccatattg gacctttaaa aacacttggg gcacggattg 2400gggagaggac ggatttttca gggtacaaca aaacataaac gcctgtggta tgagaaacga 2460acttgcgtct actgcagtca tttattaatc tcaacacact cgctatttgg aacataatca 2520tatcgtctca gtagctcaag gtagagcgta gcgctctgga tcgtatagat cttgctaagg 2580ttgtgagttc aagtctcgcc tgagatatta aaaaactttg taattttaaa aattttattt 2640tataatatac aattaaaaac tatacaattt tttattatta cattaataat gatacaattt 2700ttattattac atttaatatt gtctattacg gtttctaatc atacagtaca aaaataaaat 2760cacaattaat ataattacaa agttaactac atgaccaaac atgaacgaag tcaatttagc 2820ggccaattcg ccttcagcca tggaagtgat atcgctcaga ctggtgccga cgccgccaaa 2880cttggtgttc tccatggtgg ttatgaggtt gcttttttgt tgggcaataa acgaccagcc 2940gctggcatct ttccaactgt cgtgataggt cgtgttgccg atggtcggga tccaaaactc 3000gacgtcgtcg tcaattgcta gttccttgta gttgctaaaa tctatgcatt gcgacgagtc 3060cgtgttggcc acccaacgcc cttctttgta gatgctgttg ttgtagcaat tactggtgtg 3120tgccggcgga ttggtgcacg gcatcagcaa aaacgtgtcg tccgacaaaa atgttgaaga 3180aacagagttg ttcatgagat tgccaatcaa acgctcgtcc accttggcca cggagactat 3240caggtcgtgc agcatattgt ttagcttgtt gatgtgcgca tgcatcagct caatgttcat 3300tttcagcaaa tcgttttcgt acatcagctc ctcttgaata tgcatcaggt cgcctttggt 3360ggcagtgtct ccctctgtgt acttggctct aacgttgtgg cgccaagtgg gcggccgctt 3420cttgactcgg tgctcgactt tgcgtttaat gcatctgtta aacttgcagt tccacgtgtt 3480tttagaaaga tcatatatat cattgtcaat caaacagtgt tcgcgtgtca ccgactcggg 3540gttatttttg tcatctttaa tgagcagaca cgcagctttt atttggcgcg tggtgaacgt 3600agacttttgt ttgagaatca tactcacgcc gtctcgatga agcacagtgt ccacggtcac 3660gttgatgggg ttgccctcag cgtccaaaat gtatacctgg cactcgtccg tgtcgtcctg 3720gcactcgagc ctgctgtaca ttttcgaagt ggaaatgccg catcgccacg atttgttgca 3780cgtgtggtgc gcaaagtgat tgttattctg ccgcttcacc aactctttgc ctttgaccca 3840ctggccgcgg ccctcgttgt cgcgaaaaca gtcgtcgctg tcactgcccc aacggtcgat 3900cagctcttcg cccacctcgc actgctgcct gatgctccac ataagcaaat cctctttgcc 3960cacattcagc gttttcatgg tttcttcgac gcgtgtgttg ggatccagcg agccgccgtt 4020gtacgcatac gcctggtagt accccttgta gccgataatc acgttttcgt tgtagtccgt 4080ctccacgatg gtgatttcca cgtccttttg cagcgtttcc ttgggcgggg taatgtccaa 4140gtttttaatc ttgtacggac ccgtcttcat ttgcgcgttg cagtgctccg ccgcaaaggc 4200agaatgcgcc gccgccgcca aaagcacata taaaacaata gcgcttacca tcttgctaat 4260cccgcggcca tggcggccgg gagcatgcga cgtcgggccc aattcgccct atagtgagtc 4320gtattacaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac 4380ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc 4440ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatgga cgcgccctgt 4500agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc 4560agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc 4620tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg 4680cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga 4740tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc 4800caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg 4860ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt 4920aacaaaatat taacgcttac aatttcctga tgcggtattt tctccttacg catctgtgcg 4980gtatttcaca ccgcatcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt 5040ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg 5100cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt 5160cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta 5220aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc 5280ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa 5340gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc 5400cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt 5460acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact 5520gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac 5580aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata 5640ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta 5700ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg 5760gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat 5820aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt 5880aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga 5940aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa 6000gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag 6060gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac 6120tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc 6180gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat 6240caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat 6300actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct 6360acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt 6420cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg 6480gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta 6540cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg 6600gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg 6660tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc 6720tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg 6780gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat 6840aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc 6900agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg 6960cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt 7020gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt 7080atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac 7140agctatgacc atgattacgc caagctattt aggtgacact atagaatact caagctatgc 7200atccaacgcg ttgggagctc tcccatatgg tcgacctgca ggcggccgca ctagtgatta 7260tgggtttgtt tgcgtgttgc acaaaataca caaggctgtc gaccgacaca aaaatgaagt 7320ttccctatgt tgcgttgtcg tacatcaacg tgacgctgtg cacctacacc gccatgttgg 7380tgggatacat ggtaacattc aatgactcca gcgaattgaa atatttacaa tactggttgc 7440tgttgtcgtt tttgatgtcc gtggtgctaa acgctccgac tctgtggacg atgctcaaaa 7500ccacagaagc ccatgaagta atttacgaaa tgaagctgtt ccacgccatg tactttagta 7560acgtgctgtt gaattatgtg gtgtttttgg acaatcaaat gggtacaaat tttgtttttg 7620ttaacaattt aattcactgt tgtgtacttt ttatgatatt tgttgaattg cttatcctgt 7680tgggccacac aatgggcacg tacacggatt atcaatatgt caaatcgtgt tatatggtta 7740tattgtttgt ttcagttatg agtgttacta ttgttatggg tttagagtgt ttgaaaacga 7800aactaattga taacagtttg atgtttaacg cgtttgtgtg cgctttgtac attgtgattg 7860caataatgtg gtctttaaaa aataatttga ctagttatta cgtttcaaat ttacaaagta 7920ttcaagttgt tccgttttca tacaacgatc cgccgccacc gttctctaac attgtaatgg 7980atgacataaa aaataaaaaa taatttataa aaatgttttt tattctttca caattctgta 8040aattctaaac aaaaaatata aatacaaact tattatgttg tcgtctaaat aaacatcaat 8100ttgtaaatct ggacacctat tcatatcatt gatattacag tctactatac aacaattaaa 8160actaaccaaa ttatctttac aacaattaaa gcaattaaaa caatttaaat aatcttcatt 8220gtcgtcgtat aagtttattt gcactgtaga cggtgttaca cagcgatcca ttcgacgttc 8280gtgttcgatc aactttctcg ccaacttgta ccataaaaat tgtttggaca aaaagttttc 8340caacaatggt aacggccaat tcaacgtgac gatgcgcacg tcctcgggta tgcatttgtt 8400aaaaaacaca cagctcgctt taccaaacga aagcaaaggt actaaatatg gcgccattgg 8460ctgatttgtt attccaagat aattacaaat aaactgatcc gtcgtggggt gataactggc 8520aggtgtcagc tttaaataat cttcaacgtt gttgtcgcgc aaaagtctgc attttacacg 8580cgttgttaat cccacgactt ttgcatgtaa aatcggatcc aaatactgca gaatcgtgtc 8640tataatttct aatggtaaac gtatgcgttt tgctcgtggg cgctttgtaa cgctcgacat 8700cctaataaca actaacacaa aactaaaatg atactcaata tattgctttt acagttcatc 8760tttaggttta aactgtgcgt ttatcgcgtt gagcaagtcg ccgttatcgg catcaatctc 8820ccaagcaaac aggccgccca atttatttcg gtcgacatat ttaacttttc ctaacacaga 8880gtcgacgctg tcaaacgaaa tcaaatcacc tttactttta tcgaaaacgt acgacgcttg 8940agcggcgctg tcaaacgtgt acacataatt gttgagatct ttttgaattt gacgataatc 9000tacaacaccg tcctcccacg tgcccgaccc cggcccgttg ccagtgccgg aaaaatagtt 9060gtcattcgta taatttgtta cgccggtcca gccgcggccg tacatggcga cgcccacaat 9120tattttgttg ggatcgacgc cttgtttcag taacgcatcg acagcgtagt gtgtagtgta 9180tagctcttcc gagttccaac ttggcgcgta gactgttgtt tggtagccca aatccgtgtt 9240tgaccaagcc cctttaaaat cgtaactcat gagaaatatt ttgcctaatg acttttgcgc 9300ttcggcgtag tttaccacgg caatcttgtc gtaacccgcg cttatagcgc ttgttaattc 9360gtaaaccctg ccggtttgcg cttcgaggtc gtctagcatt gcgcgcagct cctccaacaa 9420caaaatgtat gttttggcgt caccgtccgc atcgcccaac gacgggttag cccctttgcc 9480gcccggaaac tcccaatcga tgtctacacc gtcaaagaat ttccacactt gcagaaattc 9540cttaaccgaa tctacaaaaa cgtttctttt ttcaacatcg tgcataaaat aaaatgggtc 9600tgatagagtc cagcctccta ttgaaggaag aatttttaaa tgggggtttg ctaattttgc 9660cgccatcaac tgtccaaaat tgcctttata cggctcgttc caagcggaca cacctttttg 9720gggtttttgt acggcggccc acggatcgtg aatggcaact ttgaaatctt cgcgtccctt 9780gcacgatctt tgcaaagatt caaagctgat cctccccagc atgcctgcta ttgtcttccc 9840aatcctcccc cttgctgtcc tgccccaccc caccccccag aatagaatga cacctactca 9900gacaatgcga tgcaatttcc tcattttatt aggaaaggac agtgggagtg gcaccttcca 9960gggtcaagga aggcacgggg gaggggcaaa caacagatgg ctggcaacta gaaggcacag 10020tcgaggcggg cccttactcc tctctaacac gagattccca atcggacagg atggccctca 10080gtgattgttt gatagtgatt tcgggtttcc atccagtagt atcatggagg cgggcatgag 10140agcctctaac acgacgttgc tcggcacgcc tcattctggc gggatcttga acgatttcca 10200gctcaacttg agcaatatcg gccaacagct cgatgagttc acgaattttt tgttcttgtc 10260cagagcacac gttgtaaacg gctccggcct ctccgtggga caacagtctc aggtaagcag 10320acagcacgtc ttgaacgtcc aagaagtccc tcgacacgtc gatgtcacca acttcgaggc 10380ggtttgcctg gaggccttgt ttcatcctag caatttggcg agcagcagaa gcgatcacga 10440acgaatcctt ttggccagga ccaatgtggt tgaaaggacg ggcaaccaaa acacgccagc 10500cctcagtgat tccccattgg agacacagag attcagcagc caatttggac acagcgtaag 10560ggttacgggg atgagggatg agttcctcgt gaataggcaa ggcagcctcg gccacttgac 10620cgtacacatc accggaggag atgtacagga aggttccgga gaatcctcta gccttcagag 10680cttggagcaa gttcagtgtt cccagcaggt tgatttggag agtacgagca ggatcacgga 10740atgcctcggg tacgtatgtt tgaccagcga ggtgaataac agcatccggc aattcgggcc 10800acaaatcgcc caaagagtcg ggttccaaca agtcgtagcg gtgaggaaca gggagcaaag 10860cccaaggtgt gtgagcagcc gccaagtatg cctggaggtg tttaccgacg aagccagaca 10920gtcccgttac gaacaagcgt tgagtcatgg ttcgcgaggt cacttggttg ttcacgatct 10980tgtcgccgcc agtgtcaact tgcaactgaa acaatatcca acatgaacgt caatttatac 11040tgccctaatg gcgaacacga taacaatatt tcttttatta tgccctctaa aaccaacgcg 11100gttatcgttt atttattcaa attagatata gaacatccgc cgacatacaa tgttaatgca 11160aaaacgcgtt tggtgagcgg atacgaaaac agtcggccga taaacattaa tctgaggtcg 11220ataacaccgt ccttgaacgg aacacgagga gcgtacgtga tcagctgcat tcgcgcgccg 11280cgcctttatc gagatttatt tgcatacaac aagtacactg cgccgttggg atttgtggta 11340acgcgcacac atgcagagct gcaagtgtgg cacattttgt ctgtgcgcaa aacctttgaa 11400gccaaaagta cgaggtccgt tacgggcatg ctagcgcaca cggacaatgg acccgacaaa 11460ttctacgcca aggatttaat gataatgtcg ggcaacgtat ccgttcattt tatcaataac 11520ctacaaaaat gtcgcgcgca tcacaaagac atcgacgcgc gtagaattct acccgtaaag 11580cgagtttagt tatgagccat gtgcaaaaca tgacatcagc ttttattttt ataacaaatg 11640acatcatttc ttgattgtgt tttacacgta gaattctact cgtaaagccg agagttcagt 11700tttgaaaaac aaatgacatc atctttttga ttgtgcttta cgagtagaat tctacccgta 11760aatcaagttc ggttttgaaa aacaaatgag tcatattgta tgatatcata ttgcaaaaca 11820aatgactcat caatcgatcg tgcg 11844212420DNAArtificial SequencepVL1393-polh-antiCD20-IgG 2agcgcccgat ggtgggacgg tatgaataat ccggaatatt tataggtttt tttattacaa 60aactgttacg aaaacagtaa aatacttatt tatttgcgag atggttatca ttttaattat 120ctccatgatg ctagctgagt ttcaaattgg taattggacc cttcattaag atttcacaca 180gatcagccga ctgcgaatag aaactcacct aggcactagt ctcgagatca tggagataat 240taaaatgata accatctcgc aaataaataa gtattttact gttttcgtaa cagttttgta 300ataaaaaaac ctataaatat tccggattat tcataccgtc ccaccatcgg gcgctgttta 360aacaccatgg gctggtccct gatcctgctg ttcctggtgg ccgtggccac ccgcgtgctg 420agccaggtgc agctgcagca gcccggcgcc gagctggtga agcccggcgc ctccgtgaag 480atgagctgca aggccagcgg ctacaccttc acctcctaca atatgcactg ggtgaagcag 540accccgggcc gcggcctgga gtggatcggc gccatctacc cgggcaacgg cgatacctcc 600tacaaccaga agttcaaggg caaggccacc ctgaccgccg ataagagctc cagcaccgcc 660tacatgcagc tgtcctcgct gaccagcgag gacagcgccg tgtactactg cgcccgcagc 720acctactacg gcggcgattg gtacttcaac gtgtggggcg ccggcaccac cgtgaccgtg 780agcgccgcca gcaccaaggg cccctccgtg ttcccgctgg ccccgtcgag caagagcacc 840agcggcggca ccgccgccct gggctgcctg gtgaaggatt acttcccgga gcccgtgacc 900gtgtcgtgga acagcggcgc cctgaccagc ggcgtgcaca ccttcccagc cgtgctgcag 960agctcgggcc tgtactcgct gagcagcgtg gtgaccgtgc cgtcgagctc gctgggcacc 1020cagacctaca tctgcaacgt gaaccacaag ccatccaata ccaaggtgga taagaaggcc 1080gagcccaaga gctgcgacaa gacccacacc tgccccccct gcccggcccc agagctgctg 1140ggcggcccat ccgtgttcct gttccccccg aagccgaagg acaccctgat gatcagccgc 1200acccccgagg tgacctgcgt ggtggtggat gtgagccacg aggaccccga ggtgaagttc 1260aactggtacg tggatggcgt ggaggtgcac aacgccaaga ccaagccccg cgaggagcag 1320tacaacagca cctaccgcgt ggtgtcggtg ctgaccgtgc tgcaccagga ttggctgaac 1380ggcaaggagt acaagtgcaa ggtgtccaac aaggccctgc ccgccccgat cgagaagacc 1440atctccaagg ccaagggcca gccacgcgag ccgcaggtgt acaccctgcc accctcccgc 1500gatgagctga ccaagaacca ggtgagcctg acctgcctgg tgaagggctt ctacccctcg 1560gatatcgccg tggagtggga gagcaacggc cagccggaga acaactacaa gaccacccca 1620cccgtgctgg acagcgacgg cagcttcttc ctgtacagca agctgaccgt ggacaagtcg 1680cgctggcagc agggcaacgt gttctcctgc agcgtgatgc acgaggccct gcacaaccac 1740tacacccaga agagcctgag cctgagcccc ggcaagtaac ctgcagatct gcctcgactg 1800tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg 1860aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga 1920gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg 1980aagacaatag caggcatgct ggggaggatc tgatcctttc ctgggacccg gcaagaacca 2040aaaactcact ctcttcaagg aaatccgtaa tgttaaaccc gacacgatga agcttgtcgt 2100tggatggaaa ggaaaagagt tctacaggga aacttggacc cgcttcatgg aagacagctt 2160ccccattgtt aacgaccaag aagtgatgga tgttttcctt gttgtcaaca tgcgtcccac 2220tagacccaac cgttgttaca aattcctggc ccaacacgct ctgcgttgcg accccgacta 2280tgtacctcat gacgtgatta ggatcgtcga gccttcatgg gtgggcagca acaacgagta 2340ccgcatcagc ctggctaaga agggcggcgg ctgcccaata atgaaccttc actctgagta 2400caccaactcg ttcgaacagt tcatcgatcg tgtcatctgg gagaacttct acaagcccat 2460cgtttacatc ggtaccgact ctgctgaaga ggaggaaatt ctccttgaag tttccctggt 2520gttcaaagta aaggagtttg caccagacgc acctctgttc actggtccgg cgtattaaaa 2580cacgatacat tgttattagt acatttatta agcgctagat tctgtgcgtt gttgatttac 2640agacaattgt tgtacgtatt ttaataattc attaaattta taatctttag ggtggtatgt 2700tagagcgaaa atcaaatgat tttcagcgtc tttatatctg aatttaaata ttaaatcctc 2760aatagatttg taaaataggt ttcgattagt ttcaaacaag ggttgttttt ccgaaccgat 2820ggctggacta tctaatggat tttcgctcaa cgccacaaaa cttgccaaat cttgtagcag 2880caatctagct ttgtcgatat tcgtttgtgt tttgttttgt aataaaggtt cgacgtcgtt 2940caaaatatta tgcgcttttg tatttctttc atcactgtcg ttagtgtaca attgactcga 3000cgtaaacacg ttaaataaag cttggacata tttaacatcg ggcgtgttag ctttattagg 3060ccgattatcg tcgtcgtccc aaccctcgtc

gttagaagtt gcttccgaag acgattttgc 3120catagccaca cgacgcctat taattgtgtc ggctaacacg tccgcgatca aatttgtagt 3180tgagcttttt ggaattattt ctgattgcgg gcgtttttgg gcgggtttca atctaactgt 3240gcccgatttt aattcagaca acacgttaga aagcgatggt gcaggcggtg gtaacatttc 3300agacggcaaa tctactaatg gcggcggtgg tggagctgat gataaatcta ccatcggtgg 3360aggcgcaggc ggggctggcg gcggaggcgg aggcggaggt ggtggcggtg atgcagacgg 3420cggtttaggc tcaaatgtct ctttaggcaa cacagtcggc acctcaacta ttgtactggt 3480ttcgggcgcc gtttttggtt tgaccggtct gagacgagtg cgattttttt cgtttctaat 3540agcttccaac aattgttgtc tgtcgtctaa aggtgcagcg ggttgaggtt ccgtcggcat 3600tggtggagcg ggcggcaatt cagacatcga tggtggtggt ggtggtggag gcgctggaat 3660gttaggcacg ggagaaggtg gtggcggcgg tgccgccggt ataatttgtt ctggtttagt 3720ttgttcgcgc acgattgtgg gcaccggcgc aggcgccgct ggctgcacaa cggaaggtcg 3780tctgcttcga ggcagcgctt ggggtggtgg caattcaata ttataattgg aatacaaatc 3840gtaaaaatct gctataagca ttgtaatttc gctatcgttt accgtgccga tatttaacaa 3900ccgctcaatg taagcaattg tattgtaaag agattgtctc aagctccgca cgccgataac 3960aagccttttc atttttacta cagcattgta gtggcgagac acttcgctgt cgtcgacgta 4020catgtatgct ttgttgtcaa aaacgtcgtt ggcaagcttt aaaatattta aaagaacatc 4080tctgttcagc accactgtgt tgtcgtaaat gttgtttttg ataatttgcg cttccgcagt 4140atcgacacgt tcaaaaaatt gatgcgcatc aattttgttg ttcctattat tgaataaata 4200agattgtaca gattcatatc tacgattcgt catggccacc acaaatgcta cgctgcaaac 4260gctggtacaa ttttacgaaa actgcaaaaa cgtcaaaact cggtataaaa taatcaacgg 4320gcgctttggc aaaatatcta ttttatcgca caagcccact agcaaattgt atttgcagaa 4380aacaatttcg gcgcacaatt ttaacgctga cgaaataaaa gttcaccagt taatgagcga 4440ccacccaaat tttataaaaa tctattttaa tcacggttcc atcaacaacc aagtgatcgt 4500gatggactac attgactgtc ccgatttatt tgaaacacta caaattaaag gcgagctttc 4560gtaccaactt gttagcaata ttattagaca gctgtgtgaa gcgctcaacg atttgcacaa 4620gcacaatttc atacacaacg acataaaact cgaaaatgtc ttatatttcg aagcacttga 4680tcgcgtgtat gtttgcgatt acggattgtg caaacacgaa aactcactta gcgtgcacga 4740cggcacgttg gagtatttta gtccggaaaa aattcgacac acaactatgc acgtttcgtt 4800tgactggtac gcggcgtgtt aacatacaag ttgctaaccg gcggttcgta atcatggtca 4860tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga 4920agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg 4980cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc 5040caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac 5100tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata 5160cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa 5220aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct 5280gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa 5340agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 5400cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca 5460cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa 5520ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg 5580gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg 5640tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg 5700acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc 5760tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag 5820attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac 5880gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc 5940ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag 6000taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt 6060ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag 6120ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca 6180gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact 6240ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca 6300gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg 6360tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc 6420atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg 6480gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca 6540tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt 6600atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc 6660agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc 6720ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca 6780tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa 6840aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat 6900tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa 6960aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa 7020accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc 7080gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca 7140gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt 7200ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac 7260catatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgccat 7320tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta 7380cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt 7440tcccagtcac gacgttgtaa aacgacggcc agtgccaagc tttactcgta aagcgagttg 7500aaggatcata tttagttgcg tttatgagat aagattgaaa gcacgtgtaa aatgtttccc 7560gcgcgttggc acaactattt acaatgcggc caagttataa aagattctaa tctgatatgt 7620tttaaaacac ctttgcggcc cgagttgttt gcgtacgtga ctagcgaaga agatgtgtgg 7680accgcagaac agatagtaaa acaaaaccct agtattggag caataatcga tttaaccaac 7740acgtctaaat attatgatgg tgtgcatttt ttgcgggcgg gcctgttata caaaaaaatt 7800caagtacctg gccagacttt gccgcctgaa agcatagttc aagaatttat tgacacggta 7860aaagaattta cagaaaagtg tcccggcatg ttggtgggcg tgcactgcac acacggtatt 7920aatcgcaccg gttacatggt gtgcagatat ttaatgcaca ccctgggtat tgcgccgcag 7980gaagccatag atagattcga aaaagccaga ggtcacaaaa ttgaaagaca aaattacgtt 8040caagatttat taatttaatt aatattattt gcattcttta acaaatactt tatcctattt 8100tcaaattgtt gcgcttcttc cagcgaacca aaactatgct tcgcttgctc cgtttagctt 8160gtagccgatc agtggcgttg ttccaatcga cggtaggatt aggccggata ttctccacca 8220caatgttggc aacgttgatg ttacgtttat gcttttggtt ttccacgtac gtcttttggc 8280cggtaatagc cgtaaacgta gtgccgtcgc gcgtcacgca caacaccgga tgtttgcgct 8340tgtccgcggg gtattgaacc gcgcgatccg acaaatccac cactttggca actaaatcgg 8400tgacctgcgc gtcttttttc tgcattattt cgtctttctt ttgcatggtt tcctggaagc 8460cggtgtacat gcggtttaga tcagtcatga cgcgcgtgac ctgcaaatct ttggcctcga 8520tctgcttgtc cttgatggca acgatgcgtt caataaactc ttgtttttta acaagttcct 8580cggttttttg cgccaccacc gcttgcagcg cgtttgtgtg ctcggtgaat gtcgcaatca 8640gcttagtcac caactgtttg ctctcctcct cccgttgttt gatcgcggga tcgtacttgc 8700cggtgcagag cacttgagga attacttctt ctaaaagcca ttcttgtaat tctatggcgt 8760aaggcaattt ggacttcata atcagctgaa tcacgccgga tttagtaatg agcactgtat 8820gcggctgcaa atacagcggg tcgccccttt tcacgacgct gttagaggta gggcccccat 8880tttggatggt ctgctcaaat aacgatttgt atttattgtc tacatgaaca cgtatagctt 8940tatcacaaac tgtatatttt aaactgttag cgacgtcctt ggccacgaac cggacctgtt 9000ggtcgcgctc tagcacgtac cgcaggttga acgtatcttc tccaaattta aattctccaa 9060ttttaacgcg agccattttg atacacgtgt gtcgattttg caacaactat tgttttttaa 9120cgcaaactaa acttattgtg gtaagcaata attaaatatg ggggaacatg cgccgctaca 9180acactcgtcg ttatgaacgc agacggcgcc ggtctcggcg caagcggcta aaacgtgttg 9240cgcgttcaac gcggcaaaca tcgcaaaagc caatagtaca gttttgattt gcatattaac 9300ggcgattttt taaattatct tatttaataa atagttatga cgcctacaac tccccgcccg 9360cgttgactcg ctgcacctcg agcagttcgt tgacgccttc ctccgtgtgg ccgaacacgt 9420cgagcgggtg gtcgatgacc agcggcgtgc cgcacgcgac gcacaagtat ctgtacaccg 9480aatgatcgtc gggcgaaggc acgtcggcct ccaagtggca atattggcaa attcgaaaat 9540atatacagtt gggttgtttg cgcatatcta tcgtggcgtt gggcatgtac gtccgaacgt 9600tgatttgcat gcaagccgaa attaaatcat tgcgattagt gcgattaaaa cgttgtacat 9660cctcgctttt aatcatgccg tcgattaaat cgcgcaatcg agtcaagtga tcaaagtgtg 9720gaataatgtt ttctttgtat tcccgagtca agcgcagcgc gtattttaac aaactagcca 9780tcttgtaagt tagtttcatt taatgcaact ttatccaata atatattatg tatcgcacgt 9840caagaattaa caatgcgccc gttgtcgcat ctcaacacga ctatgataga gatcaaataa 9900agcgcgaatt aaatagcttg cgacgcaacg tgcacgatct gtgcacgcgt tccggcacga 9960gctttgattg taataagttt ttacgaagcg atgacatgac ccccgtagtg acaacgatca 10020cgcccaaaag aactgccgac tacaaaatta ccgagtatgt cggtgacgtt aaaactatta 10080agccatccaa tcgaccgtta gtcgaatcag gaccgctggt gcgagaagcc gcgaagtatg 10140gcgaatgcat cgtataacgt gtggagtccg ctcattagag cgtcatgttt agacaagaaa 10200gctacatatt taattgatcc cgatgatttt attgataaat tgaccctaac tccatacacg 10260gtattctaca atggcggggt tttggtcaaa atttccggac tgcgattgta catgctgtta 10320acggctccgc ccactattaa tgaaattaaa aattccaatt ttaaaaaacg cagcaagaga 10380aacatttgta tgaaagaatg cgtagaagga aagaaaaatg tcgtcgacat gctgaacaac 10440aagattaata tgcctccgtg tataaaaaaa atattgaacg atttgaaaga aaacaatgta 10500ccgcgcggcg gtatgtacag gaagaggttt atactaaact gttacattgc aaacgtggtt 10560tcgtgtgcca agtgtgaaaa ccgatgttta atcaaggctc tgacgcattt ctacaaccac 10620gactccaagt gtgtgggtga agtcatgcat cttttaatca aatcccaaga tgtgtataaa 10680ccaccaaact gccaaaaaat gaaaactgtc gacaagctct gtccgtttgc tggcaactgc 10740aagggtctca atcctatttg taattattga ataataaaac aattataaat gctaaatttg 10800ttttttatta acgatacaaa ccaaacgcaa caagaacatt tgtagtatta tctataattg 10860aaaacgcgta gttataatcg ctgaggtaat atttaaaatc attttcaaat gattcacagt 10920taatttgcga caatataatt ttattttcac ataaactaga cgccttgtcg tcttcttctt 10980cgtattcctt ctctttttca tttttctcct cataaaaatt aacatagtta ttatcgtatc 11040catatatgta tctatcgtat agagtaaatt ttttgttgtc ataaatatat atgtcttttt 11100taatggggtg tatagtaccg ctgcgcatag tttttctgta atttacaaca gtgctatttt 11160ctggtagttc ttcggagtgt gttgctttaa ttattaaatt tatataatca atgaatttgg 11220gatcgtcggt tttgtacaat atgttgccgg catagtacgc agcttcttct agttcaatta 11280caccattttt tagcagcacc ggattaacat aactttccaa aatgttgtac gaaccgttaa 11340acaaaaacag ttcacctccc ttttctatac tattgtctgc gagcagttgt ttgttgttaa 11400aaataacagc cattgtaatg agacgcacaa actaatatca caaactggaa atgtctatca 11460atatatagtt gctgatatct ccccagcatg cctgctattg tcttcccaat cctccccctt 11520gctgtcctgc cccaccccac cccccagaat agaatgacac ctactcagac aatgcgatgc 11580aatttcctca ttttattagg aaaggacagt gggagtggca ccttccaggg tcaaggaagg 11640cacgggggag gggcaaacaa cagatggctg gcaactagaa ggcacagtcg aggcgaattc 11700ttagcactcg ccgcggttga aggacttggt caccgggctc gacaggccct ggtgggtcac 11760ctcgcaggcg tacaccttgt gcttctcgta atcggccttg gacagggtca gggtggagct 11820cagcgagtag gtgctatcct tggaatcctg ctcggtcacg ctctcctggg agttgccgga 11880ctgcagggcg ttatccacct tccactgcac cttggcctcg cgggggtaga agttgttcag 11940caggcacacc acggaggcgg tgccggactt cagctgctcg tccgacggcg ggaagatgaa 12000cacggagggg gcggccacgg tgcgcttgat ctccagcttg gtgccgccgc cgaaggtcgg 12060ggggttgctg gtccactgct ggcagtagta ggtggcggca tcctcggcct ccacgcggga 12120gatggtcagg ctgtaggagg tgccgctgcc gctgccggag aagcgcaccg gcacgccgga 12180ggccagattg gaggtggcgt agatccaggg cttcggggag ctgcctggct tctgctggaa 12240ccagtgaatg tagctcacgc tggaggaggc gcggcaggtc atggtcacct tctcgcccgg 12300cgaggcgctc aggatggcgg ggctctgcga cagcacgatc tggccgcggc tcatgatcac 12360ggaggcgctg atcagcagga aggagatgat ctgcacctgg aaatccatgg tggcggccgc 12420312812DNAArtificial SequencepVL1393-p6.9-antiCD20-IgG 3tccatggtgg cggccgcgtt taaattgtgt aatttatgta gctgtaattt ttaccttatt 60aatatttttt acgctttgca ttcgacgact gaactcccaa atatatgttt aactcgtctt 120ggtcgtttga atttttgttg ctgtgtttcc taatattttc catcacctta aatatgttat 180tgtaatcctc aatgttgaac ttgcaattgg acacggcata gttttccata gtcgtgtaaa 240acatggtatt ggctgcattg taatacatcc gactgagcgg gtacggatct atgtgtttga 300gcagcctgtt caaaaactct gcatcgtcgc aaaacggaat ttgctagctg agtttcaaat 360tggtaattgg acccttcatt aagatttcac acagatcagc cgactgcgaa tagaaactca 420cctaggcact agtctcgaga aattccgttt tgcgacgatg cagagttttt gaacaggctg 480ctcaaacaca tagatccgta cccgctcagt cggatgtatt acaatgcagc caataccatg 540ttttacacga ctatggaaaa ctatgccgtg tccaattgca agttcaacat tgaggattac 600aataacatat ttaaggtgat ggaaaatatt aggaaacaca gcaacaaaaa ttcaaacgac 660caagacgagt taaacatata tttgggagtt cagtcgtcga atgcaaagcg taaaaaatat 720taataaggta aaaattacag ctacataaat tacacaattt aaacgtttaa acaccatggg 780ctggtccctg atcctgctgt tcctggtggc cgtggccacc cgcgtgctga gccaggtgca 840gctgcagcag cccggcgccg agctggtgaa gcccggcgcc tccgtgaaga tgagctgcaa 900ggccagcggc tacaccttca cctcctacaa tatgcactgg gtgaagcaga ccccgggccg 960cggcctggag tggatcggcg ccatctaccc gggcaacggc gatacctcct acaaccagaa 1020gttcaagggc aaggccaccc tgaccgccga taagagctcc agcaccgcct acatgcagct 1080gtcctcgctg accagcgagg acagcgccgt gtactactgc gcccgcagca cctactacgg 1140cggcgattgg tacttcaacg tgtggggcgc cggcaccacc gtgaccgtga gcgccgccag 1200caccaagggc ccctccgtgt tcccgctggc cccgtcgagc aagagcacca gcggcggcac 1260cgccgccctg ggctgcctgg tgaaggatta cttcccggag cccgtgaccg tgtcgtggaa 1320cagcggcgcc ctgaccagcg gcgtgcacac cttcccagcc gtgctgcaga gctcgggcct 1380gtactcgctg agcagcgtgg tgaccgtgcc gtcgagctcg ctgggcaccc agacctacat 1440ctgcaacgtg aaccacaagc catccaatac caaggtggat aagaaggccg agcccaagag 1500ctgcgacaag acccacacct gccccccctg cccggcccca gagctgctgg gcggcccatc 1560cgtgttcctg ttccccccga agccgaagga caccctgatg atcagccgca cccccgaggt 1620gacctgcgtg gtggtggatg tgagccacga ggaccccgag gtgaagttca actggtacgt 1680ggatggcgtg gaggtgcaca acgccaagac caagccccgc gaggagcagt acaacagcac 1740ctaccgcgtg gtgtcggtgc tgaccgtgct gcaccaggat tggctgaacg gcaaggagta 1800caagtgcaag gtgtccaaca aggccctgcc cgccccgatc gagaagacca tctccaaggc 1860caagggccag ccacgcgagc cgcaggtgta caccctgcca ccctcccgcg atgagctgac 1920caagaaccag gtgagcctga cctgcctggt gaagggcttc tacccctcgg atatcgccgt 1980ggagtgggag agcaacggcc agccggagaa caactacaag accaccccac ccgtgctgga 2040cagcgacggc agcttcttcc tgtacagcaa gctgaccgtg gacaagtcgc gctggcagca 2100gggcaacgtg ttctcctgca gcgtgatgca cgaggccctg cacaaccact acacccagaa 2160gagcctgagc ctgagccccg gcaagtaacc tgcagatctg cctcgactgt gccttctagt 2220tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact 2280cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat 2340tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc 2400aggcatgctg gggaggatct gatcctttcc tgggacccgg caagaaccaa aaactcactc 2460tcttcaagga aatccgtaat gttaaacccg acacgatgaa gcttgtcgtt ggatggaaag 2520gaaaagagtt ctacagggaa acttggaccc gcttcatgga agacagcttc cccattgtta 2580acgaccaaga agtgatggat gttttccttg ttgtcaacat gcgtcccact agacccaacc 2640gttgttacaa attcctggcc caacacgctc tgcgttgcga ccccgactat gtacctcatg 2700acgtgattag gatcgtcgag ccttcatggg tgggcagcaa caacgagtac cgcatcagcc 2760tggctaagaa gggcggcggc tgcccaataa tgaaccttca ctctgagtac accaactcgt 2820tcgaacagtt catcgatcgt gtcatctggg agaacttcta caagcccatc gtttacatcg 2880gtaccgactc tgctgaagag gaggaaattc tccttgaagt ttccctggtg ttcaaagtaa 2940aggagtttgc accagacgca cctctgttca ctggtccggc gtattaaaac acgatacatt 3000gttattagta catttattaa gcgctagatt ctgtgcgttg ttgatttaca gacaattgtt 3060gtacgtattt taataattca ttaaatttat aatctttagg gtggtatgtt agagcgaaaa 3120tcaaatgatt ttcagcgtct ttatatctga atttaaatat taaatcctca atagatttgt 3180aaaataggtt tcgattagtt tcaaacaagg gttgtttttc cgaaccgatg gctggactat 3240ctaatggatt ttcgctcaac gccacaaaac ttgccaaatc ttgtagcagc aatctagctt 3300tgtcgatatt cgtttgtgtt ttgttttgta ataaaggttc gacgtcgttc aaaatattat 3360gcgcttttgt atttctttca tcactgtcgt tagtgtacaa ttgactcgac gtaaacacgt 3420taaataaagc ttggacatat ttaacatcgg gcgtgttagc tttattaggc cgattatcgt 3480cgtcgtccca accctcgtcg ttagaagttg cttccgaaga cgattttgcc atagccacac 3540gacgcctatt aattgtgtcg gctaacacgt ccgcgatcaa atttgtagtt gagctttttg 3600gaattatttc tgattgcggg cgtttttggg cgggtttcaa tctaactgtg cccgatttta 3660attcagacaa cacgttagaa agcgatggtg caggcggtgg taacatttca gacggcaaat 3720ctactaatgg cggcggtggt ggagctgatg ataaatctac catcggtgga ggcgcaggcg 3780gggctggcgg cggaggcgga ggcggaggtg gtggcggtga tgcagacggc ggtttaggct 3840caaatgtctc tttaggcaac acagtcggca cctcaactat tgtactggtt tcgggcgccg 3900tttttggttt gaccggtctg agacgagtgc gatttttttc gtttctaata gcttccaaca 3960attgttgtct gtcgtctaaa ggtgcagcgg gttgaggttc cgtcggcatt ggtggagcgg 4020gcggcaattc agacatcgat ggtggtggtg gtggtggagg cgctggaatg ttaggcacgg 4080gagaaggtgg tggcggcggt gccgccggta taatttgttc tggtttagtt tgttcgcgca 4140cgattgtggg caccggcgca ggcgccgctg gctgcacaac ggaaggtcgt ctgcttcgag 4200gcagcgcttg gggtggtggc aattcaatat tataattgga atacaaatcg taaaaatctg 4260ctataagcat tgtaatttcg ctatcgttta ccgtgccgat atttaacaac cgctcaatgt 4320aagcaattgt attgtaaaga gattgtctca agctccgcac gccgataaca agccttttca 4380tttttactac agcattgtag tggcgagaca cttcgctgtc gtcgacgtac atgtatgctt 4440tgttgtcaaa aacgtcgttg gcaagcttta aaatatttaa aagaacatct ctgttcagca 4500ccactgtgtt gtcgtaaatg ttgtttttga taatttgcgc ttccgcagta tcgacacgtt 4560caaaaaattg atgcgcatca attttgttgt tcctattatt gaataaataa gattgtacag 4620attcatatct acgattcgtc atggccacca caaatgctac gctgcaaacg ctggtacaat 4680tttacgaaaa ctgcaaaaac gtcaaaactc ggtataaaat aatcaacggg cgctttggca 4740aaatatctat tttatcgcac aagcccacta gcaaattgta tttgcagaaa acaatttcgg 4800cgcacaattt taacgctgac gaaataaaag ttcaccagtt aatgagcgac cacccaaatt 4860ttataaaaat ctattttaat cacggttcca tcaacaacca agtgatcgtg atggactaca 4920ttgactgtcc cgatttattt gaaacactac aaattaaagg cgagctttcg taccaacttg 4980ttagcaatat tattagacag ctgtgtgaag cgctcaacga tttgcacaag cacaatttca 5040tacacaacga cataaaactc gaaaatgtct tatatttcga agcacttgat cgcgtgtatg 5100tttgcgatta cggattgtgc aaacacgaaa actcacttag cgtgcacgac ggcacgttgg 5160agtattttag tccggaaaaa attcgacaca caactatgca cgtttcgttt gactggtacg 5220cggcgtgtta acatacaagt tgctaaccgg cggttcgtaa tcatggtcat agctgtttcc 5280tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg 5340taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc 5400cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg 5460gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc 5520ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac 5580agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa 5640ccgtaaaaag gccgcgttgc tggcgttttt

ccataggctc cgcccccctg acgagcatca 5700caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc 5760gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata 5820cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta 5880tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 5940gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga 6000cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg 6060tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg 6120tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg 6180caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 6240aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa 6300cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat 6360ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc 6420tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc 6480atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc 6540tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc 6600aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc 6660catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt 6720gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc 6780ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa 6840aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt 6900atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg 6960cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc 7020gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa 7080agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt 7140gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt 7200caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag 7260ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta 7320tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat 7380aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat 7440catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg 7500tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta 7560agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg 7620gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg 7680tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgccatt cgccattcag 7740gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc 7800gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg 7860acgttgtaaa acgacggcca gtgccaagct ttactcgtaa agcgagttga aggatcatat 7920ttagttgcgt ttatgagata agattgaaag cacgtgtaaa atgtttcccg cgcgttggca 7980caactattta caatgcggcc aagttataaa agattctaat ctgatatgtt ttaaaacacc 8040tttgcggccc gagttgtttg cgtacgtgac tagcgaagaa gatgtgtgga ccgcagaaca 8100gatagtaaaa caaaacccta gtattggagc aataatcgat ttaaccaaca cgtctaaata 8160ttatgatggt gtgcattttt tgcgggcggg cctgttatac aaaaaaattc aagtacctgg 8220ccagactttg ccgcctgaaa gcatagttca agaatttatt gacacggtaa aagaatttac 8280agaaaagtgt cccggcatgt tggtgggcgt gcactgcaca cacggtatta atcgcaccgg 8340ttacatggtg tgcagatatt taatgcacac cctgggtatt gcgccgcagg aagccataga 8400tagattcgaa aaagccagag gtcacaaaat tgaaagacaa aattacgttc aagatttatt 8460aatttaatta atattatttg cattctttaa caaatacttt atcctatttt caaattgttg 8520cgcttcttcc agcgaaccaa aactatgctt cgcttgctcc gtttagcttg tagccgatca 8580gtggcgttgt tccaatcgac ggtaggatta ggccggatat tctccaccac aatgttggca 8640acgttgatgt tacgtttatg cttttggttt tccacgtacg tcttttggcc ggtaatagcc 8700gtaaacgtag tgccgtcgcg cgtcacgcac aacaccggat gtttgcgctt gtccgcgggg 8760tattgaaccg cgcgatccga caaatccacc actttggcaa ctaaatcggt gacctgcgcg 8820tcttttttct gcattatttc gtctttcttt tgcatggttt cctggaagcc ggtgtacatg 8880cggtttagat cagtcatgac gcgcgtgacc tgcaaatctt tggcctcgat ctgcttgtcc 8940ttgatggcaa cgatgcgttc aataaactct tgttttttaa caagttcctc ggttttttgc 9000gccaccaccg cttgcagcgc gtttgtgtgc tcggtgaatg tcgcaatcag cttagtcacc 9060aactgtttgc tctcctcctc ccgttgtttg atcgcgggat cgtacttgcc ggtgcagagc 9120acttgaggaa ttacttcttc taaaagccat tcttgtaatt ctatggcgta aggcaatttg 9180gacttcataa tcagctgaat cacgccggat ttagtaatga gcactgtatg cggctgcaaa 9240tacagcgggt cgcccctttt cacgacgctg ttagaggtag ggcccccatt ttggatggtc 9300tgctcaaata acgatttgta tttattgtct acatgaacac gtatagcttt atcacaaact 9360gtatatttta aactgttagc gacgtccttg gccacgaacc ggacctgttg gtcgcgctct 9420agcacgtacc gcaggttgaa cgtatcttct ccaaatttaa attctccaat tttaacgcga 9480gccattttga tacacgtgtg tcgattttgc aacaactatt gttttttaac gcaaactaaa 9540cttattgtgg taagcaataa ttaaatatgg gggaacatgc gccgctacaa cactcgtcgt 9600tatgaacgca gacggcgccg gtctcggcgc aagcggctaa aacgtgttgc gcgttcaacg 9660cggcaaacat cgcaaaagcc aatagtacag ttttgatttg catattaacg gcgatttttt 9720aaattatctt atttaataaa tagttatgac gcctacaact ccccgcccgc gttgactcgc 9780tgcacctcga gcagttcgtt gacgccttcc tccgtgtggc cgaacacgtc gagcgggtgg 9840tcgatgacca gcggcgtgcc gcacgcgacg cacaagtatc tgtacaccga atgatcgtcg 9900ggcgaaggca cgtcggcctc caagtggcaa tattggcaaa ttcgaaaata tatacagttg 9960ggttgtttgc gcatatctat cgtggcgttg ggcatgtacg tccgaacgtt gatttgcatg 10020caagccgaaa ttaaatcatt gcgattagtg cgattaaaac gttgtacatc ctcgctttta 10080atcatgccgt cgattaaatc gcgcaatcga gtcaagtgat caaagtgtgg aataatgttt 10140tctttgtatt cccgagtcaa gcgcagcgcg tattttaaca aactagccat cttgtaagtt 10200agtttcattt aatgcaactt tatccaataa tatattatgt atcgcacgtc aagaattaac 10260aatgcgcccg ttgtcgcatc tcaacacgac tatgatagag atcaaataaa gcgcgaatta 10320aatagcttgc gacgcaacgt gcacgatctg tgcacgcgtt ccggcacgag ctttgattgt 10380aataagtttt tacgaagcga tgacatgacc cccgtagtga caacgatcac gcccaaaaga 10440actgccgact acaaaattac cgagtatgtc ggtgacgtta aaactattaa gccatccaat 10500cgaccgttag tcgaatcagg accgctggtg cgagaagccg cgaagtatgg cgaatgcatc 10560gtataacgtg tggagtccgc tcattagagc gtcatgttta gacaagaaag ctacatattt 10620aattgatccc gatgatttta ttgataaatt gaccctaact ccatacacgg tattctacaa 10680tggcggggtt ttggtcaaaa tttccggact gcgattgtac atgctgttaa cggctccgcc 10740cactattaat gaaattaaaa attccaattt taaaaaacgc agcaagagaa acatttgtat 10800gaaagaatgc gtagaaggaa agaaaaatgt cgtcgacatg ctgaacaaca agattaatat 10860gcctccgtgt ataaaaaaaa tattgaacga tttgaaagaa aacaatgtac cgcgcggcgg 10920tatgtacagg aagaggttta tactaaactg ttacattgca aacgtggttt cgtgtgccaa 10980gtgtgaaaac cgatgtttaa tcaaggctct gacgcatttc tacaaccacg actccaagtg 11040tgtgggtgaa gtcatgcatc ttttaatcaa atcccaagat gtgtataaac caccaaactg 11100ccaaaaaatg aaaactgtcg acaagctctg tccgtttgct ggcaactgca agggtctcaa 11160tcctatttgt aattattgaa taataaaaca attataaatg ctaaatttgt tttttattaa 11220cgatacaaac caaacgcaac aagaacattt gtagtattat ctataattga aaacgcgtag 11280ttataatcgc tgaggtaata tttaaaatca ttttcaaatg attcacagtt aatttgcgac 11340aatataattt tattttcaca taaactagac gccttgtcgt cttcttcttc gtattccttc 11400tctttttcat ttttctcctc ataaaaatta acatagttat tatcgtatcc atatatgtat 11460ctatcgtata gagtaaattt tttgttgtca taaatatata tgtctttttt aatggggtgt 11520atagtaccgc tgcgcatagt ttttctgtaa tttacaacag tgctattttc tggtagttct 11580tcggagtgtg ttgctttaat tattaaattt atataatcaa tgaatttggg atcgtcggtt 11640ttgtacaata tgttgccggc atagtacgca gcttcttcta gttcaattac accatttttt 11700agcagcaccg gattaacata actttccaaa atgttgtacg aaccgttaaa caaaaacagt 11760tcacctccct tttctatact attgtctgcg agcagttgtt tgttgttaaa aataacagcc 11820attgtaatga gacgcacaaa ctaatatcac aaactggaaa tgtctatcaa tatatagttg 11880ctgatatctc cccagcatgc ctgctattgt cttcccaatc ctcccccttg ctgtcctgcc 11940ccaccccacc ccccagaata gaatgacacc tactcagaca atgcgatgca atttcctcat 12000tttattagga aaggacagtg ggagtggcac cttccagggt caaggaaggc acgggggagg 12060ggcaaacaac agatggctgg caactagaag gcacagtcga ggcgaattct tagcactcgc 12120cgcggttgaa ggacttggtc accgggctcg acaggccctg gtgggtcacc tcgcaggcgt 12180acaccttgtg cttctcgtaa tcggccttgg acagggtcag ggtggagctc agcgagtagg 12240tgctatcctt ggaatcctgc tcggtcacgc tctcctggga gttgccggac tgcagggcgt 12300tatccacctt ccactgcacc ttggcctcgc gggggtagaa gttgttcagc aggcacacca 12360cggaggcggt gccggacttc agctgctcgt ccgacggcgg gaagatgaac acggaggggg 12420cggccacggt gcgcttgatc tccagcttgg tgccgccgcc gaaggtcggg gggttgctgg 12480tccactgctg gcagtagtag gtggcggcat cctcggcctc cacgcgggag atggtcaggc 12540tgtaggaggt gccgctgccg ctgccggaga agcgcaccgg cacgccggag gccagattgg 12600aggtggcgta gatccagggc ttcggggagc tgcctggctt ctgctggaac cagtgaatgt 12660agctcacgct ggaggaggcg cggcaggtca tggtcacctt ctcgcccggc gaggcgctca 12720ggatggcggg gctctgcgac agcacgatct ggccgcggct catgatcacg gaggcgctga 12780tcagcaggaa ggagatgatc tgcacctgga aa 12812431DNAArtificial Sequenceprimer 4gtttaaacgc ctcgactgtg ccttctagtt g 31526DNAArtificial Sequenceprimer 5actagttccc cagcatgcct gctatt 26634DNAArtificial Sequenceprimer 6aatggatccg gtaccaccat ggtgagcaag ggcg 34736DNAArtificial Sequenceprimer 7ggcacttcgc gattacttgt acagctcgtc catgcc 36833DNAArtificial Sequenceprimer 8ttatctcgcg aaccatgact caacgcttgt tcg 33926DNAArtificial Sequenceprimer 9atcacggaat gcctcgggta cgtatg 261026DNAArtificial Sequenceprimer 10gctggtcaaa catacgtacc cgaggc 261134DNAArtificial Sequenceprimer 11tgggccctta ctcctctcta acacgagatt ccca 341235DNAArtificial Sequenceprimer 12attgtgaatt cgcctcgact gtgccttcta gttgc 351332DNAArtificial Sequenceprimer 13taattgatat ctccccagca tgcctgctat tg 321451DNAArtificial Sequenceprimer 14gccgcgctag catcatggag ataattaaaa tgataaccat ctcgcaaata a 511532DNAArtificial Sequenceprimer 15aatttgcggc cgcagcgccc gatggtggga cg 321650DNAArtificial Sequenceprimer 16gggcctcgag atcatggaga taattaaaat gataaccatc tcgcaaataa 501737DNAArtificial Sequenceprimer 17atttgcatgc gtttaaacag cgcccgatgg tgggacg 371832DNAArtificial Sequenceprimer 18gccgcgctag caaattccgt tttgcgacga tg 321951DNAArtificial Sequenceprimer 19aatttgcggc cgcgtttaaa ttgtgtaatt tatgtagctg taatttttac c 512031DNAArtificial Sequenceprimer 20gggcctcgag aaattccgtt ttgcgacgat g 312156DNAArtificial Sequenceprimer 21atttgcatgc gtttaaacgt ttaaattgtg taatttatgt agctgtaatt tttacc 562229DNAArtificial Sequenceprimer 22atttgtattt aatcaatcga accgtgcac 292330DNAArtificial Sequenceprimer 23gattgggaat ctcgtgttag agaggagtaa 302424DNAArtificial Sequenceprimer 24atcttcttca aggacgacgg caac 242534DNAArtificial Sequenceprimer 25agcaagatgg taagcgctat tgttttatat gtgc 342630DNAArtificial Sequenceprimer 26agatgggtat gaaaccatac aacaagtgtg 302728DNAArtificial Sequenceprimer 27cgctaccata atctttgttg aatcgatg 28

Claims

1. A recombinant baculovirus expression vector for transient expression of GDP-4-dehydro-6-deoxy-D-mannose reductase (RMD) in an insect cell, said vector comprising the following operably linked components, i) an expression control sequence functional early in infection operably linked to a codon optimized RMD encoding nucleic acid; and ii) an insertion site suitable for insertion of one or more nucleic acids encoding at least one heterologous protein of interest.

2. The recombinant baculovirus expression vector of claim 1, which is AcRMD.

3. The recombinant baculovirus expression vector of claim 1 or claim 2 comprising a nucleic acid sequence encoding a heterologous protein of interest operably linked to a promoter active later in infection. inserted at said insertion site.

4. The recombinant baculovirus expression vector of claim 1, wherein said expression control sequence is selected from the group consisting of a constitutive promoter and an inducible promoter.

5. The recombinant baculovirus expression vector of claim 4, wherein said constitutive promoter is a baculovirus immediate early promoter selected from the group consisting of ie1, ie2, ie0, et1, and gp64,insect actin, tubulin, a ubiquitin promoter; RSV promoter, copia, gypsy promoter and a cytomegalovirus IE promoter.

6. The recombinant baculovirus expression vector of claim 4, wherein said inducible promoter is selected from the group consisting of baculovirus delayed early, late, and very late promoters, an hsp70 promoter, a metallothionein promoter and a tetracycline-regulated promoter.

7. The recombinant baculovirus expression vector of claim 3, wherein said nucleic acid encoding said at least one heterologous protein of interest comprises a promoter selected from the group consisting of a promoter from baculovirus delayed early, late, and very late promoters.

8. The recombinant baculovirus expression vector of claim 4, wherein said expression control sequence comprises a promoter and an enhancer element that increases activity of said promoter.

9. The recombinant baculovirus expression vector of claim 3, wherein said protein of interest is a therapeutic protein.

10. The recombinant baculovirus expression vector of claim 9, wherein said protein of interest is selected from the group consisting of an antibody, a subunit vaccine, an antibiotic, a cytokine, an anticoagulant, a viral antigen, an enzyme, a hormone, and a blood clotting factor.

11. An infected insect cell comprising the recombinant baculoviral vector of claim 1 or claim 3, selected from the group consisting of Sf9, Sf21, expresSF+.RTM., Tn368, High Five.RTM., Tni PRO.RTM., Ea4, Ao38, BmN, S2, and S2R+.

12. A method for producing at least one molecule of interest lacking fucose, comprising: a) providing insect cells; b) introducing a baculovirus comprising at least one nucleic acid molecule encoding the enzyme GDP-4-dehydro-6-deoxy-D-mannose reductase (RMD) operably driven by an immediate early expression control sequence for expression immediately after infection or an inducible promoter, thereby stabilizing inhibition of fucosylation, and at least one additional nucleic acid molecule encoding at least one heterologous protein of interest driven by an promoter active later in infection, thereby producing non-fucosylated proteins wherein said additional nucleic acid is present on the same baculovirus encoding RMD or is present on a second baculovirus vector; c) incubating under conditions wherein GDP-4-dehydro-6-deoxy-D-mannose reductase blocks the production of GDP-L-fucose, and said at least one protein of interest is produced lacking fucose; and d) isolating said at least one protein of interest.

13. The method of claim 12, wherein said RMD enzyme and said protein of interest are encoded by a single recombinant baculoviral vector and expressed sequentially at earlier and later times of infection.

14. The method of claim 12, wherein said nucleic acid encoding said enzyme and said protein of interest are on separate baculovirus vectors and expressed sequentially at earlier and later times of infection.

15. The method of claim 12, wherein said protein of interest is a therapeutic protein.

16. The method of claim 15, wherein said therapeutic protein is selected from the group consisting of an antibody, a subunit vaccine, an antibiotic, a cytokine, an anticoagulant, a viral antigen, an enzyme, a hormone, and a blood clotting factor.

17. The method of claim 16, wherein said therapeutic protein is an antibody.

18. A kit for the production of at least one protein of interest lacking fucose or with a reduced amount of fucose comprising at least one recombinant baculovirus comprising at least one nucleic acid molecule encoding the enzyme GDP-4-dehydro-6-deoxy-D-mannose reductase (RMD) operably driven by an immediate early expression control sequence for expression early after infection, thereby stabilizing inhibition of fucosylation, and an insertion site for at least one additional nucleic acid molecule encoding at least one protein of interest operably driven by a control sequence for expression later in infection for production of non-fucosylated proteins of interest.

19. The kit of claim 18, further comprising insect cells.

20. The kit of claim 18, further comprising a second baculoviral vector comprising a promoter suitable to drive expression of said protein of interest later in in infection and an insertion site for insertion of a nucleic acid encoding said protein of interest. 21. The kit of claim 18, wherein said protein of interest is selected from the group consisting of an antibody, cytokine, blood clotting factor, anticoagulant, viral antigen, enzyme, receptor, vaccine, subunit vaccine, and hormone.

22. A method for production of a non-fucosylated protein in insect larvae, comprising a) providing insect larvae; b) introducing a baculovirus comprising at least one nucleic acid molecule encoding the enzyme GDP-4-dehydro-6-deoxy-D-mannose reductase (RMD) operably driven by an immediate early expression control sequence for expression immediately after infection or an inducible promoter, thereby stabilizing inhibition of fucosylation, and at least one additional nucleic acid molecule encoding at least one protein of interest driven by an expression control sequence active later in infection, thereby producing non-fucosylated proteins wherein said additional nucleic acid is present on the same baculovirus encoding RMD or is present on a second baculovirus vector; c) incubating under conditions wherein GDP-4-dehydro-6-deoxy-D-mannose reductase blocks the production of GDP-L-fucose, and said at least one protein of interest is produced lacking fucose; and d) isolating said protein of interest.

23. The recombinant baculovirus vector of claim 8, wherein said enhancer is hr5.