Evaluation of novel control elements by construction of eukaryotic expression vectors

Gene 188 (1997) 191–198

Evaluation of novel control elements by construction of eukaryotic expression vectors Christina Furebring a, Mats Ohlin a, Sven Pettersson b, Carl A.K. Borrebaeck a,* a Department of Immunotechnology, Lund University, P.O. Box 7031, S-220 07 Lund, Sweden b Center for Biotechnology, Karolinska Institute, Huddinge, Sweden Received 8 July 1996; revised 7 October 1996; accepted 12 October 1996; Received by C.M. Kane

Abstract A novel mammalian eukaryotic expression vector for the production of immunoglobulin heavy chain (IgH ) genes has been designed. This expression vector contains the variable heavy chain ( VH ) promoter, the IgH intron enhancer (mE ) and the IgH 3∞ enhancer (3∞E ). This construct, designated pTIF-1, was stably transfected into the myeloma cell line J558L. A fivefold increase in the expression level of a rearranged IgH gene was observed when using the pTIF-1 vector containing the 3∞E compared to an expression vector lacking this enhancer. Interestingly, this positive effect on the expression level of the 3∞ enhancer appears to be position independent. The introduction of two recently identified Ig control elements, HS3 and HS4, to the vector cassette did not further elevate the expression level in the cell line tested. The pTIF-1 vector can be used for expression of any antibody specificity, using PCR amplification of the VDJ region of interest. Furthermore, the constant region can easily be exchanged, which further facilitates studies to dissect different effector functions of IgH constant genes. Keywords: Antibody; 3∞ Enhancer; Intron enhancer; Hypersensitive sites 3 and 4

1. Introduction Immunoglobulin molecules are unique in that a wide range of binding specificities is coupled to different effector functions localized in a constant molecular framework. These characteristics make them potentially very useful in a variety of therapeutic regimes. Hybridomas are one main source of antibodies but since most of these antibodies have been of murine origin, they have induced an anti-immunoglobulin response when administered in vivo. Recombinant DNA technology has, however, made it possible to produce chimeric, humanized and human antibodies or fragments thereof, using either prokaryotic or eukaryotic expression systems. Since effector functions mediated by the immunoglobulin (Ig) constant region are required in most * Corresponding author. e-mail: [email protected] Abbreviations: Cc3, human constant c3 region; bp, base pair(s); 3∞E, 3∞ mouse Ig heavy chain enhancer; HS3, HS4, hypersensitive sites 3, 4 DNA element; Ig, immunoglobulin; kb, kilobase(s) or 1000 bp; mE, mouse Ig heavy chain intron enhancer; NP, 4-hydroxy3-nitrophenylacetyl; PBS, phosphate buffered saline; SDB, sample dilution buffer; VH, variable heavy chain region.

therapeutic regimes and since prokaryotic hosts are unable to produce these structures in a functional form, these molecules must be produced in mammalian cells (Shin et al., 1992). Today, several different vectors are available for both transient and stable expression, the latter being most appropriate for large scale production (Adair, 1992; Gillies, 1992). Expression of Ig genes within the lymphoid lineage was for a long time thought to be regulated only by the VH promoter and the intron enhancer (mE ). Many eukaryotic expression vectors use these control elements, although the level of produced antibodies are, in the majority of cases, lower than what has been obtained from hybridoma cell lines (Liu et al., 1987; Queen et al., 1989; Sun et al., 1987). Recently, new enhancer elements, like the 3∞aE, 3∞E, HS3 and HS4, have been identified that might contribute to a higher expression of the Ig gene. The 3∞aE is localized in close vicinity to the Ca gene and is a quite weak potentiator (Matthias and Baltimore, 1993). One of the most studied elements is the 3∞ heavy chain enhancer (3∞E), which is localized 16 kb downstream from the IgH locus in mice (Pettersson et al., 1990; Dariavach et al., 1991). This enhancer is located in a region where two DNase I

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hypersensitive sites have been identified and the 3∞E can be divided into three functionally independent domains, controlled by different DNA binding proteins like Ets, Pax, Oct and AP-1 proteins (Grant et al., 1992, 1995; Neurath et al., 1995; Giannini et al., 1993). In recent transgenic animal experiments we have indeed shown that the expression level was higher if the transgene was controlled by both the mE and the 3∞E, as compared to transgenes controlled by the mE alone (Arulampalam et al., 1996), indicating that there are probably additional elements involved in the regulation of Ig gene expression. Recently, two new DNase I hypersensitive sites have been localized 13 kb (HS3) and 17 kb (HS4) further downstream from the 3∞E. It has been shown that these three elements together conferred positionindependent, copy-number-dependent expression of a cmyc gene stably integrated into a plasmacytoma cell line (Madisen and Groudine, 1994; Michaelson et al., 1995). In this report, we have employed these control elements in a novel mammalian expression vector, in order to evaluate their ability to direct high levels of Ig gene expression.

2. Materials and methods 2.1. Initial cloning of enhancer elements 2.1.1. b-Globin construct A basic vector for expression of b-globin under the control of its endogenous promoter (Pettersson et al., 1990) was used in these cloning experiments. The 3∞E (1 kb) fragment was isolated from genomic mouse DNA (Dariavach et al., 1991) as a StuI fragment and cloned upstream or downstream from the b-globin gene. The 3∞E (4 kb) fragment was similarly isolated from genomic mouse DNA (Dariavach et al., 1991) and cloned either as a HindIII (upstream) or as a XbaI (downstream) fragment into the b-globin vector. The different constructs are depicted in Fig. 1. 2.1.2. Ig gene expression vector constructs To allow efficient enhancer element cloning into various Ig gene encoding vectors, suitable fragments were prepared and cloned as outlined in Fig. 3. Briefly, the mE was isolated from genomic mouse DNA as a XbaI fragment (Banerij et al., 1983) and cloned into the Bluescript M13+ vector (construct BV.1). The 3∞E (1 kb) was PCR amplified from genomic mouse DNA and cloned into the pSP72 vector as a HindIII-XhoI fragment (construct D.1). The 3∞E (4 kb) was available in the 3∞E-pIC2OH vector (construct C.1) (Dariavach et al., 1991). The HS3 enhancer element was PCR amplified from mouse genomic DNA and cloned as a HindIII fragment into the 3∞E/pSP72 vector (construct D.2). Similarly, the HS4 enhancer element was PCR

amplified and cloned as a XbaI fragment into the 3∞E/HS3/pSP72 vector (construct D.3). All primers used for PCR amplification are outlined in Table 1. 2.2. Construction of the backbone eukaryotic expression vector The human genomic Cc3 gene was isolated from a pUC19 vector, kindly provided by Dr. I. Sandlie (Oslo, Norway), and cloned as a HindIII-BamHI fragment into the mE/Bluescript M13+ vector (construct BV.2). The VH promoter was first cloned as a HindIII fragment into the pSP72 vector and then recloned into the mE/Cc3/Bluescript vector as a SalI-XhoI fragment (construct BV.3). The VH gene, encoding the variable heavy chain domain of a NP-specific antibody, was amplified from the hybridoma B1-8-d1 (Pettersson et al., 1990) using leader sequence and FR4-specific primers ( Table 1), and cloned as a ClaI fragment. To create the backbone fragment a NotI site was inserted at the KpnI site of the vector using an oligonucleotide linker (Stratagene, La Jolla, CA) permitting the isolation of the backbone fragments (containing the VH promoter, the VH gene, the c3 constant gene and the mE) as a NotI fragment (construct BV ). 2.3. Construction of expression vectors with different control elements The basic construction of the various Ig heavy chain gene expression vectors is outlined in Figs. 3 and 4. Briefly, the pmE-VH vector was constructed by cloning the backbone as a NotI fragment into the pIF2 vector, which contains the selectable neo marker (construct A). To prepare the p3∞E(1 kb)-VH vector, the 3∞E (1 kb) fragment was recloned as a XhoI-SalI fragment, from Table 1 Primers used for polymerase chain reaction-base amplification of antibody variable region genes and enhancer elements Leader sequence primer: CTC GCA GAG ATC GAT ATG GGA TGG AGC TGT ATC FR4 primer: TCA TTC GCT ATC GAT ACT CAC CTG AGG AGA CTG TGA G 3∞E primers: CCG TAT AAG CTT CTG TCT CCA CGT GGC CAC CCG GCG CTC GAG GCC TGT CTC CAT GTG GCC HS3 primers: GCT AAG CTT ACC ACA TGC GAT CTA AGG GAT ATT GGG GCA TGG AAG CTT ATT GAG CTC CGG CTC TAA CAA CTG GGT CCT HS4 primers: GCT TCT AGA GGA GTT AGG TGG GTA GGT GAG TGC AGG CAC TGG TCT AGA ACT CAC TGT TCA CCA TGA ACC CAG CTA GTC

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the 3∞E/pSP72 vector into the backbone vector. Thereafter the whole insert was cloned into the pIF2 vector as a NotI fragment (construct B). The construction of the p3∞E(4 kb)-VH vector was carried out following introduction of a NotI site at the ClaI site of the 3∞E-pIC2OH vector using an oligonucleotide linker. The backbone fragment was subsequently cloned into this vector as a NotI fragment (construct C ). Finally, the p(3∞E/HS3/HS4)-VH vector was made by introducing a NotI site at the ClaI site of the 3∞E/HS3/HS4/pSP72 vector using an oligonucleotide linker permitting cloning of the backbone fragment into this vector (construct D). 2.4. Transfection and transient expression of the b-globin gene The murine B cell line MPC-11 was used for transfection and was grown in Dulbecco’s modified Eagle’s medium, containing 10% fetal calf serum, penicillin and streptomycin (Gibco, Grand Island, NY ). MPC-11 cells in log phase were transfected with 20 mg of the linearized plasmid DNA, using a calcium phosphate coprecipitation method (Graham and van der Eb, 1973). In each transfection, 5 mg of control vector expressing the human a -globin gene, using the SV40 enhancer was 2 included ( Weston, 1988). Cells were harvested for preparation of cytoplasmic RNA 30–36 h after transfection. 2.5. Transfection and stable expression of immunoglobulin genes 15 mg of DNA of either expression vector was linearized by PvuI digestion and 7 mg of DNA for the neo gene vector was linearized by BamHI digestion. 6.5×106 J558L cells (a spontaneous heavy-chain-lossvariant myeloma cell line) were used for each transfection. This cell line expresses a l light chain which in combination with the heavy chain gene employed in this study will form a NP-specific complete antibody. Prior to transfection the cells were washed with cold PBS and then resuspended in 0.65 ml of the same cold buffer and preincubated 10 min with the linearized expression vector. When transfecting the p3∞E(4 kb)-VH (construct C ) or the p(3∞E/HS3/HS4)-VH (construct D) vectors, 7 mg of BamHI-linearized pIF2 vector was cotransfected as it carries the selectable neo marker. The mixture was placed in a 1.9 mm electrode gap electroporation cuvette (BTX Inc., San Diego, CA) into the Electro Cell Manipulator 200 (BTX Inc). Two square pulses with an amplitude of 600 V and a duration of 99 ms were applied. After the pulses the cells were incubated on ice for 10 min followed by addition of RPMI 1640 (Gibco, Grand Island, NY ) supplemented with 10% fetal calf serum, 4 mM -glutamine and 50 mg gentamicin/ml. The transfected cells were plated into 96-well microtiter plates at a concentration of 104 cells/well. The selective

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agent G418 (Gibco) was added 48 h later at a final concentration of 2 mg/ml. Approx. 2 weeks after addition of selection medium the culture supernatants were screened by ELISA to test for the secretion of heavy chain. High producers were selected, expanded and then subcloned at limiting dilution. Five clones were selected from each transfection. Each clone was cultured for 3 days starting with 50 000 cells/ml, and samples were taken every 12 h and the cells were counted. 2.6. RNAse protection assay Total cytoplasmic RNA was prepared from transfected cells and ribonuclease protection assays were carried out, as previously described (Pettersson et al., 1990). 2.7. Enzyme linked immunosorbent assay (ELISA) 96-well microtiter plates were coated overnight at 4°C with goat anti-human IgG (Zymed Lab Inc., San Fransisco, CA, USA) diluted 1:2000 in PBS. Samples were diluted, using sample dilution buffer (SDB) (10 mM sodium phosphate pH 8.0, 0.5 M NaCl, 0.1% Tween 20). Antibodies were detected, using horseradish peroxidase-conjugated goat anti-human IgG (Zymed Lab Inc.) diluted 1:2000 in SDB. The chromogenic substrate o-phenylenediamine (OPD) (Sigma Chemical, St. Louis, MO, USA) was used. For quantification of the antibody a human IgG3 antibody LuNm03 ( Ferna´ndez de Cossı´o et al., 1992) was used as a standard.

3. Results and discussion A multitude of studies have unraveled several control mechanisms governing the regulation of immunoglobulin gene expression in B-lineage cells. Key regulators are cis-acting enhancer elements (e.g. mE, 3∞aE, 3∞E, HS3 and HS4) whose activities are dictated by trans-acting transcription factors. Based on the observation that the 3∞ end of the IgH locus could act as a LCR (Madisen and Groudine, 1994), it was of interest to determine whether any of the elements located in the 3∞ end could improve productivity of recombinant antibodies in eukaryotic host cells in comparison to earlier generations of expression vectors. For this purpose various cis-acting enhancer elements were incorporated into expression vectors driven by the mouse VH promoter. 3.1. 3∞ Enhancer – size and positional effects It has been shown both in transient expression and in transgenic animals that the 3∞E can increase the amount of Ig gene expression (Dariavach et al., 1991;

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Arulampalam et al., 1996). In these transgenic animals a 1 kb StuI-StuI fragment containing the core enhancer was used. This fragment contains the two DNase I hypersensitive sites and binding sites for different transcription factors. In the transgenic studies the transgene controlled by the mE+3E did not show copy-numberdependent expression of the transgene, indicating that yet other control elements are involved in the regulation of gene expression. It is conceivable that other elements close to the 1 kb fragment of the 3∞E might be of importance. Consequently, we investigated the ability of the 4 kb fragment in addition to the 1 kb genomic fragment of the 3∞E to enhance b-globin gene transcription. It has been shown that the 3∞E can function in combination with exogenous promoters, like the b-globin promoter (Dariavach et al., 1991). The 1 kb or the 4 kb fragment was cloned either upstream from the b-globin gene or on both sides of the gene ( Fig. 1). Ribonuclease protection assays showed that the amount of RNA was somewhat higher when the b-globin gene was controlled by the 4 kb fragment as compared to the 1 kb fragment (Fig. 2). However, the level was not substantially higher indicating that no important additional control element with respect to transcriptional potentiation was found within this 4 kb fragment of the 3∞E as compared to the 1 kb fragment when analyzed in plasma cells. In addition, no difference was seen on RNA expression levels depending on whether the 3∞E (4 kb) element was cloned up- or downstream from the promoter/reporter gene (data not shown). We also analyzed whether the 3∞aE together with the 3∞E could promote expression of the b-globin gene. No further increase in gene expression was detected as compared to the expression levels achieved using only the 3∞E (data not shown). This confirmed earlier findings that the 3∞aE is only a weak potentiator of transcription (Matthias and Baltimore, 1993). Enhancer elements can in general increase transcription of a gene independently of their position or orientation relative to the promoter.

Fig. 2. Ribonuclease mapping of RNA from MPC-11 cells transfected with plasmids containing the different 3∞E fragments. The positions of full-length b-globin and a-globin (reference) nuclease-protected transcripts are indicated. A radiolabeled MspI-digested pBR322 plasmid was used to serve as a marker ( lane 1). Samples include RNA transcribed from vectors without any enhancer element ( lane 2) and with the 3∞E (1 kb) cloned upstream ( lane 3) and the 3∞E (4 kb) cloned upstream ( lane 4) from the coding sequences. Transcription from vectors carrying the 3∞E (1 kb) ( lane 5) and the 3∞E (4 kb) ( lane 6) cloned on both sides of the gene is also shown.

They can also stimulate transcription when linked to homologous as well as heterologous promoters (Moreau et al., 1981). The mE has been shown to function like these viral enhancer elements (Banerij et al., 1983; Gillies et al., 1983; Neuberger, 1983). Here we have shown that the 3∞E enhances transcription independently of position and also stimulates a heterologous promoter. Experiments using multimerized short enhancer segments Schaffner et al. (1988) showed a correlation between the number of segments and transcriptional enhancement. Therefore we cloned the 3∞E on both sides of the b-globin gene. However, no copy-number-dependent increase in transcription of the b-globin gene was observed ( Fig. 2). In conclusion, either the 4 kb or the 1 kb enhancer is likely to function in a copy-independent manner in a vector intended for use in plasma cell-like host cells. 3.2. Design of expression vectors

Fig. 1. The various b-globin constructs used for comparing gene expression controlled by different enhancer fragments in the MPC-11 cell line. A basic vector for expression of b-globin under the control of its endogenous promoter was used, the different 3∞E fragments were cloned into this vector.

A high level of antibody expression in hybridomas is well documented, but when the immunoglobulin genes are cloned into eukaryotic expression vectors and transfected into murine myeloma cell lines the expression level normally drops substantially. It is known that the VH promoter/mE pair can promote gene transcription in this system but that it is not sufficient to ensure high level expression in vitro. The chromatin structure and methylation pattern of the 3∞E changes during B cell differentiation indicating that the 3∞E is active only late in B cell development

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Fig. 3. Outline of the construction of the heavy chain vectors. The backbone vector contains the VH promoter, a VH chain (NP-specific), the human c3 constant region and the mE. Different enhancer element combinations were cloned into this vector. The completed Ig heavy chain expression vectors are the backbone fragment vector (BV ), mE/VHpro vector (A), 3∞E/mE/VHpro vector (B), 3∞E(4 kb, 5∞)/mE/VHpro vector (C ), 3∞E(1 kb, 5∞)/HS3/HS4/mE/VHpro vector (D), 3∞E(4 kb, 3∞)/mE/VHpro vector ( E ) and 3∞E(1 kb, 3∞)/HS3/HS4/mE/VHpro vector (F ).

(Giannini et al., 1993; Madisen and Groudine, 1994). It has also been shown, using an enhancer-dependent reporter gene construct in transgenic animals, that the 3∞E is mainly active in activated B cells (Arulampalam et al., 1994). Lieberson et al. (1995) have shown in a plasma cell line where the mE is deleted that Ig is still secreted whereas if the 3∞E also is abolished the IgH gene transcription ceases, indicating the importance of the 3∞E for Ig expression in cells of this differentiation stage. It has also been shown in transgenic animals that adding the 3∞E indeed increased the level of transgene Ig gene expression but no copy-dependent expression was seen, which indicates that some other elements are missing (Arulampalam et al., 1996). It has been proposed that the 3∞E together with two recently identified regions, HS3 and HS4, would function as a complete locus control region (LCR). Such a LCR would mediate copy-number-dependent Ig gene expression and recent experiments, using the myc gene as a reporter gene in plasma cell lines, support this possibility (Madisen and Groudine, 1994). Based on these observations we investigated whether these three elements could influence Ig gene expression in the same way in the light chain producing cell line J558L transfected with various heavy chain expression constructs (Fig. 4). As shown in Fig. 5, the 1 kb 3∞E could increase the Ig secretion fivefold over the levels achieved with only the mE as the enhancer element. In agreement with the observations described above, using the b-globin-expressing constructs, there

Fig. 4. The control elements of the different expression vectors used for investigating the effect of enhancers on Ig gene expression.

was no significant improvement of Ig productivity if the 4 kb 3∞E was used instead of the 1 kb 3∞E. Surprisingly, the 3∞E(1 kb)/HS3/HS4 enhancer construction in combination with mE offered no additional elevation of protein production, as compared to vectors devoid of the HS3/HS4 elements. Furthermore, Mocikat et al. (199, 1995)) reported that the distance between the mE and

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Fig. 5. Determination of Ig expression levels using vectors carrying only the mE (A), the mE and the 3∞E (1 kb) (B), the mE and the 3∞E (4 kb) 5∞ of the VH promoter (C ), all four enhancer elements (mE, 3∞E (1 kb), HS3 and HS4) 5∞ of the VH promoter (D), the 3∞E (4 kb) 3∞ of the VH promoter ( E ) and finally the four enhancer elements 3∞ of the VH promoter ( F ). The different vector constructs were transfected into the cell line J558L. After selection and cloning of the different vector constructs five clones were selected from each transfection for protein concentration analysis. The monoclonal antibody concentrations were determined after cultivation of 5×105 cells/ml for 24 h. The bars represent mean values of antibody concentration from the five different clones.

the 3∞E is of importance in their eukaryotic expression vectors. If the mE and the 3∞E are in close proximity to each other, they observed a suppression of the Ig gene expression and when the distance was increased, the gene expression increased. We also assessed whether the position of the 3∞E in relation to the mE would be of importance. However, based on our results we concluded that no increased protein expression was achieved as a consequence of modifying the position of the 3∞E(4 kb) or the 3∞E/HS3/HS4 (in either gene orientation) in relation to the VH promoter, using the J558L cell as expression host (data not shown). These results agree with the finding described above showing no positiondependent effects of the 3∞E on mRNA levels transcribed from the b-globin gene. The absence of increased transcription using the combination of 3∞E/HS3/HS4 enhancers most probably is related to the characteristics of the host cells used in these investigations. Both Singh and Birshtein (1993) and Michaelson et al. (1995) have shown that the activities of 3∞E and HS4 were dependent on host cell line. They showed that the activity of the enhancer elements was higher in the plasmacytoma cell line S194 as compared to J558L and 1165. Consequently, S194 might represent a different stage of plasmacytoma development, since it is known that the cell differentiation stage is of importance for the ability of particular regulatory elements to affect gene transcription (Neuberger, 1983). Alternatively, some of these cell lines might have lost their capacity to produce particular transacting transcription factors required for proper function of these enhancer elements. For example, certain non-Ig producing fusion partners upon fusion with normal B cells not only produce the intended B cell encoded antibody but also upregulate the production of their own endogenous Ig product. Such findings suggest

that these cell lines are deficient in certain transcription factors. We have along the same principles previously reported the spontaneous downregulation of heavy chain encoding mRNA in heterohybridoma cell lines without loss of the heavy chain encoding gene itself (Ohlin and Borrebaeck, 1994). It is thus conceivable that any aberrant genetic makeup of the cell lines selected for transfection experiments may strongly influence the outcome of antibody product yield. The Ig gene expression level achieved using the mE/VHpro construct is comparable with other reports, using the same type of construct (Nakatani et al., 1989; Queen et al., 1989; Walls et al., 1993). One alternative way to increase eukaryotic expression levels is to use a strong exogenous promoter like the CMV promoter. Bebbington et al. (1992) described one example of such a vector, using the CMV-MIE promoter/enhancer pair, which also uses glutamine synthetase as an amplifiable selectable marker for expression in myeloma cells. This vector (approx. four copies/cell ) was reported to reach its peak production at 10–15 pg/(cell×day) in exponentially growing cultures. Our vector, driving Ig gene expression through the heavy chain promoter controlled by the 3∞E and mE (3∞E/mE/VHpro), designated pTIF-1, achieved a maximal productivity, in these initial experiments, of 4–6 pg/(cell×day). The potential to increase the expression level of pTIF-1 even further involves the inclusion of amplifiable selectable markers like glutamine synthetase, and of heterologous promoter/ enhancer pairs like the CMV promoter/enhancer. In addition to the improved productivity levels of pTIF-1, as compared to vectors based on the Ig heavy chain promoter controlled by the mE alone, this vector has a number of interesting features which can be exploited for the production of recombinant antibodies. The pTIF-1 vector was designed as a general expression vector for any antibody, using the ClaI site for cloning of VH regions. We have successfully expressed the VH gene derived from the human monoclonal antibody B715A2 specific for tetanus toxoid (Malmborg et al., 1992), using the pTIF-1 vector. In these experiments, pTIF-1 was cotransfected into Sp2/0 myeloma cells together with an appropriate light chain encoding vector (pAN4621) kindly provided by Dr. S.L. Morrison (Los Angeles, CA) (Coloma et al., 1992) carrying the B715A2 VL gene (data not shown). Furthermore, this vector allows easy exchange of the heavy chain constant region as a ClaI-BamHI restriction fragment, facilitating studies of antibody effector functions.

4. Conclusions (1) The 3∞E can upregulate IgH gene expression fivefold in our eukaryotic expression vector.

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(2) The positive effect of the 3∞E on the expression level is position independent. (3) The pTIF-1 vector is designed for easy cloning of any variable heavy chain of interest. (4) The HS3 and HS4 control elements did not further enhance the expression levels.

Acknowledgement We thank Drs. Sherie Morrison and Inger Sandlie for their generous vector donations. This study was supported by the Immunotechnology Program of NUTEK and the Swedish Cancer Foundation. The skillful technical assistance by Hele´ne Turesson is gratefully acknowledged.

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