EXAMPLE 1

Cloning of the Heavy and Light Chain cDNAs for Campath-1H

[0046] The complementarity determining regions from the rat Campath-1G monoclonal were originally grafted directly into genomic human heavy and light chain frameworks (Winter et al, Nature, 1988, 322, 323-327).

[0047] These constructs were engineered for expression in the myeloma cell line YO and resulted in yields f Campath-1H of up to 5 μg/ml following 10-14 days in culture (Hale et al, Tissue Antigens, 1990, 35, 118-127 and Winter et al, Nature, 1988, 322, 323-327). The myeloma cell line TF57 (Hale et al, ibid ,) was used to generate size selected cDNA fractions of 0.9-1.2 kb and 1.4-1.7 kb for the light and heavy chain cDNAs respectively. These were used to make EcOR1 Tinkered cDNA libraries in λgt10. All procedures were as described by Huynh et al ( DNA Cloning , Vol I: A Practical Approach, 1984, Glover, D(Editor), IRL Press, oxford). The libraries were screened using [ ³² P] nick translated probes specific for the variable regions to isolate full length cDNA clones. For the light chain cDNA, the 5′ untranslated leader was removed up to position −32 using Bal-31 exonuclease and a HindIII linker added. For the 3′ end, use was made of a unique. SacI site 47 bp upstream of the stop codon. A SacI-HindIII oligonucle tide pair was used to regenerate this sequence and position the HindIII site immediately after the stop codon. For the 5′ end of the heavy chain cDNA, the unique NcoI site overlapping the ATG start codon was used to re-build a 29 bp untranslated leader, identical to that of the light chain, using a HindIII-NcoI oligonucleotide pair. At the 3′ end, the unique NaeI site 12 bp downstream of the stop codon was converted into a HindIII site using linkers.

EXAMPLE 2

Construction of Vectors

[0048] The human β-actin promoter was excised from pHβAPr-3-neo (which corresponds to pHβAPr-1-neo (Gunning et al P.N.A.S., 1987, 84, 483-35) except that the SV40 polyadenylation/termination signal has been replaced with the respective human β-actin signals) as a 2860 bp PvuII-HindIII fragment, in which the PvuII site was subsequently converted to a Bg1II site using linkers. To isolate the human β-actin polyadenylation and termination signals from pHβAPr-3-neo, an SphI site 1.4 kb downstream of the unique HindIII site was converted to a BamHI site using linkers. The basal dhfr vector called p104, was constructed as follows. The SphI site at position −128 in the SV40 promoter in pSV2 dhfr (Subramani et al, Mol, Cell, Biol, 1981, 1, 854-864) was converted into a SalI site to remove all enhancer elements from the promoter. The weakened dhfr expression unit was then subcloned as a SalI-BamHI fragment into the homologous sites in pSVOd (Mellon et al, Cell, 1981, 27, 279-288).

[0049] To construct pLD9, the p104 vector was digested with BamHI, phosphatased, and ligated with three other fragments consisting of the BglII-HindIII β-actin promoter, the HindIII Campath-1H light chain cDNA and the HindIII-BamHI β-actin polyA/termination signals. To construct pNH316, the construct pdBPV-MMTneo (Law, et al, Mol, Cell, Biol., 1983, 3, 2110-2115) was digested with BamHI, phosphatased, and the fragment containing the neomycin gene isolated following separation on an agarose gel. This was ligated to the two β-actin fragments and the Campath-1H heavy chain cDNA. The constructs, pLD9 and pNH316 are depicted in FIG. 1 .

EXAMPLE 3

Expression of Campath-1H in CHO Cells

[0050] The dhfr CHO cell line DUK-B11 (Urlaub et al, P.N.A.S., 1980, 77, 4216-4220) was grown in Iscove's MEM supplemented with 10% fetal bovine serum, and 4 μg/ml each of hypoxanthine and thymidine. 10 μg of pLD9 and pNH316 was co-precipitated onto cells using the calcium phosphate method, (Gorman et al, DNA Cloning, 1985, Vol II, 143-190, Academic Press, N.Y.) and selected for the double phenotype of dhfr ⁺ /neo resistance by using the medium above except that 10% dialysed serum was used, the hypoxanthine/thymidine were omitted, and G418 (Gibco) was included at 500 μg/ml. In some experiments MTX was included directly in the first round selection for dhfr ⁺ transformants. Several hundred resistant colonies were pooled and assayed for the production of Campath-1H antibody in the culture medium. The average yield was 0.5 μg/ml for non-amplified first round transformants.

[0051] Each pooled cell population was then cultured in the presence of 10 ⁻⁷ M MTX, and after two weeks, resistant colonies were again pooled and titred for Campath-1H production. There was a considerable increase in yield of up to 80-fold (Table 1). These cells were dilution cloned, screened for Campath-1H yield, and two high producer lines isolated, called A37 and 3D9 (Table 1). These were both amplified further in the presence of 10 ⁻⁶ M MTX, then dilution cloned and screened as above. The increase in expression ac this second, and final, amplification stage was not so dramatic as seen previously; nevertheless, when re-fed at confluence and left for a further 4 days, the cell lines A39 and 3D11 were capable of producing up to 200 μg/ml of Campath-1H. 1

[0052] Legend to Table

[0053] Cells were allowed to reach confluence in a T-175 tissue culture flask, then re-fed with fresh 50 ml of tissue culture medium and left for a further 4 days. The Campath-1H antibody that had accumulated in the medium during this period was measured by ELISA. Total cell counts on the day of assay were usually 2.5×10 ⁷ . The yield from the 3D11 cell line reflects a productivity of 100 μg/10 ⁶ cells/day.

[0054] The co-transfection vectors pLD9 and pNH316 were further employed to evaluate an alternative amplification strategy to the one described above. The dhfr ⁻ CHO cells were co-transfected as usual, and two days later split directly into a series of flasks containing G418 (for neomycin selection) and increasing concentrations of MTX ranging from 3×10 ⁻⁹ M to 10 ⁻⁷ M. Following two weeks of this selection, the number of resistant colonies were counted and pooled for each flask. When the cell populations had stabilized, they were assayed for Campath-1H antibody titres and the results are shown in Table 2. As the MTX level was increased, there was a marked decrease in the number of surviving dhfr ⁺ colonies, but they expressed proportionately more Campath-1H. Thus, in a one step direct selection at high concentrations of MTX, it is possible to isolate cell populations which produce up to 60-fold increase in antibody yield compared to cell populations selected for basal dhfr levels. 2

[0055] Legend to Table

[0056] Colonies at each MTX selection stage were pooled and assayed as described in the legend of Table 1.

[0057] This selection procedure was repeated following another co-transfection of cells, and in this instance, the entire population was selected in medium containing G418 and 3×10 ⁻⁸ M MTX. This generated a larger pool of resistant colonies which were subsequently pooled and re-amplified twice more using MTX concentrations of 6×10 ⁻⁷ M, then 3×10 ⁻⁶ M. At this stage, the cells were dilution cloned and screened for Campath-1H levels. The two highest producer cell lines isolated were capable of producing antibody levels up to 100-150 μg/ml and were designated as lines 4F11 and 5E10.

[0058] The growth rates of these cell lines, and the A39/3D11 lines described above, were considerably slower than the parental non-transformed dhfr ⁻ CHO cells. This is usually a common feature of these cells once they have been engineered to express high quantities of a product gene. The yields from the 5E10 and 4F11 cell lines proved to be quite variable over time, and the latter appeared to have only a limited passage life lasting about 3 weeks before entering crisis and death. This instability was not evident at all in the other cell lines, although in general, the lines isolated from the second amplification procedure, including 5E10, were usually more fickle to culture. Of all the lines, the 3D11 coupled good growth and stability with high Campath-1H yields. To ensure the propagation of these features, the 3D11 cell line was dilution cloned once more to generate the 3D11* line and this similarly produced Campath-1H yields up to 200 μg/ml.

EXAMPLE 4

Growth of and Production from C1H 3D11* 44 in WCM4

[0059] a) C1H 3D11* cells growing as a monolayer in Iscoves +10% FBS Flow, non-essential amino acids, 10 ⁻⁶ M Methotrexate and antibiotics were approximately 90% confluent. These cells were removed from the plastic with trypsin/versene, washed in Iscoves medium without supplements, centrifuged and resuspended at 5×10 ⁴ /ml in WCM4 medium Table 3+0.25% peptone +0.1% polyethylene glycol (PEG) 10,000+0.5% fetal boine serum (FBS) without methotrexate (MTX). 3

[0060] Three 25 cm ² flasks were set up with 10 ml of cell suspension +hypoxanthine (H), thymidine (T) or HT. These flasks were incubated at 36.5° C. in 5% CO ₂ incubator.

[0061] After six days, the flasks were pooled and added to an equal volume of WCM4+MTX without peptone or PEG, and were transferred to a 75 cm ² flask.

[0062] These cells were used to seed a 500 ml Techner spinner, incubated at 36.5° C. spinning at 40 rpm. Cells continued growing-serum free for a period of over five months and although it was found that the cells needed a period of adaptation, the growth rate and viability steadily improved. The population doubling time was calculated to be 73.1 hours over approximately 7 weeks; this decreased to 47.4 hours over the subsequent 20 days then stabilised. Antibody secretion remained high at levels in excess of 60 μg/ml. It was determined that the gene copy number in these cells did not decrease according to band intensity using Northern blot analysis.

[0063] In fermenters, these cells produced antibody in excess of 70 μg/ml and regularly achieve levels of 100 μg/ml or more. These cells are denoted C1H 3D11* 44.

[0064] b) Cells from a) above which had been growing serum-free for over 2 months were transferred to a SGi 1 litre fermenter with a stainless steel angled paddle turning at 70 rpm. The temperature was set at 37° C., dO ₂ at 10% and pH control to 7-7.2. The fermenter was seeded on day 0 with 0.22×10 ⁶ cells/ml in WCM4 (Table 3) with 0.1% polyethylene glycol (PEG) 10,000 and 0.25% soy peptone, and was top gassed with O ₂ The cells were routinely passaged using fresh medium and a split rate typically between 1 to 2 and 1 to 4.

[0065] On day 33 the top gassing was replaced with deep sparging which is can be expected to cause more physical damage to the cells.

[0066] On day 50 onwards WCM5 (Table 4) was used together with peptone and PEG instead of WCM4. 4

[0067] On day 53 the PEG was replaced with 0.1% pluronic F68. The resulting growth and antibody levels achieved are shown the the attached graphs ( FIGS. 4 and 5 ), and demonstrate the capacity of the invention to allow protein-free production of antibody in excess of 100 μg/ml in fermenters.

EXAMPLE 5

Analysis of the Rate of Campath-1H Synthesis and Secretion from CHO Cells

[0068] During the course of culturing the Campath-1H producing CHO cells of Example 3, it became clear that even when they reached confluence, antibody levels continued to accumulate, with time, in the culture medium. To determine whether this was possibly a consequence of intracellular accumulation coupled to slow secretion, the rates of Campath-1H synthesis and secretion were measured using A39 cells. These analyses were performed over 3-4 consecutive days on cells which were either in growth phase, or confluent stationary phase. For the cells in either growth state, the results were identical, and data is presented only for the immuno-precipitated radiolabelled Campath-1H produced from stationary cells.

[0069] The rate of antibody synthesis was measured by pulsing the cells for a short period with [S ³⁵ ]-methionine on each of four consecutive days, and then examining the quantity, and distribution, of immuno-precipitated material. In FIG. 2 , it is clear that the rate of synthesis is equally high at all time points measured. Furthermore, even by the end of this short pulse, in each case, more than half of the newly synthesized Campath-1H is already present in the medium suggesting rapid secretion. This was confirmed by the data shown in FIG. 3 , in which parallel cells were similarly pulsed, and the distribution of the radiolabelled Campath-1H chased over a three day period. Within 24 hours, virtually all of the cellular radiolabelled antibody has been chased into the medium, where it remained stable for the duration of the experiment. This demonstrates that even when the recombinant CHO cells remain stationary for long periods, the rates of Campath-1H synthesis and secretion are not diminished.

[0070] Campath-1H ELISA assay. Microtiter plates were coated with anti-human IgG and incubated with the assay sample (in culture medium). Antibody detection was visualized by using an anti-human gamma chain specific peroxidase conjugate.

[0071] Analysis of rates of Campath-1H synthesis and secretion. Cells from Example 3 were grown to confluence in 3 cm tissue culture wells, then incubated for 30 minutes in methionine-free Dulbeccos's MEM containing 10% fetal calf serum. Following this, the cells were labelled in the presence of 120 μCi/ml [ ³⁵ S] methionine (>800Ci/mmol; Amersham) for the appropriate time period, then either harvested and lysed in 500 μl of NP-40 lysis buffer, or incubated further in normal growth medium. Then 125 μl aliquots of cell lysate or culture medium were immuno-precipitated using goat anti-human IgG (heavy chain specific; Sigma) and 10 % protein-A Sepharose (Pharmacia). Samples were then separated on 10% SDS-PAGE reducing gels according to Laemmli and the signals amplified with Enhance (NEN-Dupont). The dried gels were then autoradiographed overnight.

[0072] Biological Assays for Functional CHO-glycosylated Campath 1H

[0073] Complement Lysis Assay for Campath 1H

[0074] The complement lysis assay is a measure of antibody function expressed as specific activity, determined by the ability of a CHO-glycosylated antibody of known concentration to bind to a pre-determined number of cells and effect cell lysis.

[0075] The assay is carried out on Campath 1H from Example 4 using Karpas 422 cells (established from B-cell non-Hodgkin lymphoma cell line—Dyer et al., (1990) Blood, 75 704-714) expressing Campath antigen on the cell surface. 1.2×10 ⁷ cells were loaded with radiolabel by incubating for 2 hours at 37° C. in a CO ₂ incubator in the presence of 600 μCi of 51Cr (sodium chromate).

[0076] 5.3 ml of the loaded cells in medium (total volume 23.5 ml), were added to 12.5 ml of normal human serum and 150 μl of the mixture were pipetted into the wells of a microtitre plate.

[0077] 50 μl samples of the final eluate from three purification runs were mixed with the cells and incubated for 30 minutes at 4° C. followed by 90 minutes at 37° C. The culture was centrifuged at 2000 rpm for 5 minutes and the radioactivity in 100 μl of cell supernatant was counted on a gamma counter. Complement lysis activity in Kilo Units/ml was calculated from a standard curve of a reference preparation (1000 Units/ml).

[0078] The results are set out in Table 5.

[0079] The concentration of Campath 1H in the 50 μl samples of final eluate was estimated using samples in PBS pH 7.2 read on a spectrophotometer at 280 nm. The results are expressed in Table 3 as optical density in mg/ml.

[0080] From this data the specific activity: 1 $\frac{\mathrm{KU}\text{/}\mathrm{ml}}{\mathrm{OD}}$

[0081] is determined. 5

[0082] The results indicate that CHO-glycosylated Campath 1H is functional.

[0083] Treatment of an Individual with CHO-glycosylated Campath 1H

[0084] An individual diagnosed as having severe T-cell mediated inflammation of the joints (immobilising polyarthritis, pleuritis, abdominal pains) over five years requiring long periods of hospitalisation was treated with CHO derived Campath 1H from Example 4 using the following regime:

[0085] 2 mg per day over 6 days by intravenous injection

[0086] 10 mg per day over subsequent 6 days by intravenous injection.

[0087] During the second 6 day treatment there was a significant symptomatic improvement. By the end of the second period the joint inflammation was much improved and a skin abscess had cleared with antibiotic treatment. Thirty days after the end of the treatment the individual was discharged.

[0088] Approximately 9 months after the initial treatment, the individual suffered a relapse with multiple joint involvement. After initial testing for sensitivity with a low dose, the individual was given a further course of treatment with 10 mg/day Campath 1H for 10 days with significant improvement.

EXAMPLE 6

[0089] Expression of Humanised ANTI-CD4 Antibody from Cho Cells

[0090] Construction of the Expression Vector pBan1: Modification of 2342-12

[0091] The complementarily determining regions from a rat IgG2b raised against human CD4 (The New England Journal of Medicine 1990 323: 250-254) were grafted onto human heavy and light chain frameworks (Winter et al, Nature, 1988, 322 323-327).

[0092] The cDNA encoding the humanised CD4 light chain was cloned into pLD9 (Page and Sydenham, M. A. 1991 Biotechnology 9 64-681. The resulting plasmid was designated p2110. The humanised CD4 heavy chain was sequenced and cloned into a modified version of plasmid p342-12 [Law M-F., Byrne, J. C. and Hinley, P. M. 1983 Mol. Cell. Biol. 3 2110-2115).

[0093] Plasmid p342-12 was digested with BamH1 to remove the 7.4 kbp fragment containing part of the BPV-1 genome. The backbone containing the β-lactamase gene and the neomycin resistance gene under the control of the mouse metallothionine promoter was purified and religated at the BamH1 site. This plasmid was digested with HinDIII, incubated with the large fragment of DNA polymerase I to remove the HinDIII site and then relegated. The β-actin expression cassette, containing the β-actin promoter immediately upstream of a unique HinDIII site followed by the polyadenylation signal, was cloned into the BamHI site of the modified p342-12 plasmid to generate pban1.

[0094] Plasmid pBan1, therefore, consisted of the neomycin resistance gene, the β-lactamase gene and the β-actin expression cassette containing the unique HinDIII site. The cDNA encoding the humanised heavy chain was cloned into this site and the resulting plasmid containing the correctly orientated insert was designated pBanCD4H. Thus, p2110 and pBanCD4H contained a different selectable marker and co-transfection into recipient dhfr-CHO cells would permit the direct selection and isolation of dhfr ⁺ /neo ^r colonies. Cells exhibiting this phenotype should express functional antiCD4 antibody and could be amplified to elevate the antibody titres.

[0095] Expression of anti-CD4 antibody in CHO cells

[0096] a) Cell culture methods.

[0097] The dhfr—CHO line DUK-B11 [Urlaub, C. and Chasin, L. A. 1980 Proc. Natl. Acad. Sci. USA 77 4216-4220] was propagated in Iscoves MEM medium supplemented with 10% foetal bovine serum and ⁴ μg each of hypoxanthine and thymidine (all Flow). After transfection, transformants were selected in the medium described above except that the hypoxanthine/thymidine were omitted and dialysed foetal bovine serum was used. In addition, G418 was included at 500 μg/ml. To induce spontaneous amplification of sequences containing and flanking the dhfr gene, MTX was added to a concentration of 0.1 μM.

[0098] b) Transfection and amplification

[0099] The dhfr—CHO cell line DUK-B11 was co-transfected with 5 μg of p2110 and 5 μg of pBanCD4H using the transfectam reagent under the conditions recommended by the manufacturer. Transformants were selected for the dhfr ⁺ /neo ^r phenotype as described above. Several hundreds of transformants were observed and pooled. Initital titres indicated that the first round basal transformants were secreting about 0.1 μg/ml/day. This po led population was then cultured in the presence of 0.1 μM MTX for about 14 days. Resistant colonies were again pooled and assayed. Expression had increased some 100 fold, the pooled, amplified colonies producing about 10-12;1g/ml/day. In order to obtain stable, clonal cell lines giving high antibody titres, the resistant pools were cloned by limiting dilution in 96-well plates. Fifty single colonies were identified and assayed and the four lines giving the highest titres propagated. This process of identifying highly expressing clones within the resistant population produced a line designated D419 which expressed the anti-CD4 antibody at about 20 μg/ml/day.

[0100] Characterisation of dhfr ⁺ /neo ^r cell lines

[0101] i) Determination of copy number and steady state transcription levels by slot blot analysis of DNA and RNA.

[0102] Whole cell RNA and DNA was prepared from the various stages of amplification as described by Maniatis et al. [1982 Molecular Cloning. A Laboratory Manual. Cold Spring Harbour Lab ratery, Cold Spring Harbour, N.Y.]. After fixing onto nitrocellulose filters, the nucleic acids were probed with [ ³² -P]-αATP labelled DNA sequences of the heavy chain, the dhfr gene and the β-actin gene as a control “housekeeping” gene to eliminate artifacts due to loading errors.

[0103] Inititally, the uncloned 0.1 μM MTX amplified pool was compared to the first round unamplified transformants and the untransformed parental B11 cells, with the probes described. Accordingly, no DNA signal was detected in the parental line when probed with the heavy chain but a weak signal was detected for dhfr. This is due to the single, non-functional dhfr allele in the B11 cell line. As a result, no RNA signal was detected with either probe. In contrast, a strong signal was detected with both probes on RNA and DNA in the primary transformants which reflects the start of expression. A very significant increase in copy number and steady state levels of RNA of heavy chain and dhfr is observed in the uncloned amplified pool. This accurately correlates with the observed increase in expression. Steady state levels of β-actin RNA were consistent in all three lines examined.

[0104] A similar comparison was made between the four highest expressing cloned cell lines. A strong signal was detected on both the RNA and the DNA blots. However, although the DA19 line was expressing twice as much antibody as a line designated D423, this difference was not in either the copy number or steady state levels of RNA. There are two possible explanations for this observation; the first is that the DNA in the DA19 line has integrated at a site in the genome at which it is under the influence of an enhancer. However, this presumably would be reflected in elevated levels of RNA. The more likely explanation is that in the replication and duplication of the tandem arrays in the line D423, some of the copies of the dhfr/antibody cassette have undergone re-arrangement and are non-functional and truncated. This is not uncommon since the site of integration of heterologous genes is often at breakpoints in the chromosomes such as telomeres which are known to be “hot spots” for such re-arrangements. This could be resolved by Northern and Southern analysis.

[0105] ii) Protein synthesis and secretion of anti-CD4 antibody in the D419 line

[0106] The clonal D419 line was labelled with ³⁵ S-methionine and cysteine and the intracellular and secreted antibody extracted by immunoprecipitation with appropriate antibodies. Following electrophoresis on reducing SDS-PAGE gels, the gels were dried and the signal detected by autoradiography.

[0107] It was clear from the result chat both heavy and light chain are efficiently synthesised. Intracellularly, there need not be stochiometry between heavy and light chains since the two associate as they pass through the secretory organelles. However, close stochiometry is observed in the secreted material.