EXPRESSION VECTOR ELEMENT COMBINATIONS, NOVEL PRODUCTION CELL GENERATION METHODS AND THEIR USE FOR THE RECOMBINANT PRODUCTION OF POLYPEPTIDES

Abstract

Herein is reported that for transient transfections the use of the human elongation factor 1 alpha promoter (with Intron A) provides for an enhanced productivity (in LC-HC-SM organization), the use of the bovine growth hormone polyA signal sequence provides for an enhanced productivity compared to use of the SV40 polyA signal sequence, the addition of the HUT to the bGH PolyA signal sequence results in an increased productivity in vectors containing the hCMV promoter and the vector organization LC(3-5)-HC-SM results in improved expression. For stable pools it is reported that pools generated with vectors containing the hEF1 promoter show an enhanced productivity in batch analysis, clones generated with vectors containing the hEF1 promoter show a reduced number of low producing clones, and clones generated with vectors containing the hEF1 promoter show a higher stability of IgG expression. For single clones it is reported that the vector organization with downstream position of selection marker (LC-HC-SM) has a positive effect on productivity of single clones and that clones generated with vectors containing the bGH polyA signal sequence and the hGT have higher productivities.

Claims

1-108. (canceled)

109. A method for selecting a recombinant mammalian cell, comprising the steps of: a) transfecting a mammalian cell with an expression vector comprising a first expression cassette comprising in 5 to 3 direction a hCMV promoter, a nucleic acid encoding an antibody light chain, a bGH polyA signal sequence, and a hGT terminator sequence, a second expression cassette comprising in 5 to 3 direction a hCMV promoter, a nucleic acid encoding an antibody heavy chain, a bGH polyA signal sequence, and a hGT terminator sequence, and and thereby obtaining a multitude of recombinant mammalian cells, and b) selecting from the multitude of recombinant mammalian cells a (single) recombinant mammalian cell.

110. The method according to claim 109, wherein the nucleic acid encoding the antibody light chain and/or the nucleic acid encoding the antibody heavy chain comprises at least one intron.

111. The method according to claim 109, wherein the nucleic acid encoding the antibody light chain and/or the nucleic acid encoding the antibody heavy chain is cDNA.

112. The method according to claim 110, wherein the nucleic acid encoding the antibody light chain and/or the nucleic acid encoding the antibody heavy chain is cDNA.

113. The method according to claim 109, wherein the first expression cassette and the second expression cassette are arranged unidirectional for the selection of a stable transfected cell.

114. The method according to claim 110, wherein the first expression cassette and the second expression cassette are arranged unidirectional for the selection of a stable transfected cell.

115. The method according to claim 111, wherein the first expression cassette and the second expression cassette are arranged unidirectional for the selection of a stable transfected cell.

116. The method according to claim 109, wherein the first expression cassette and the second expression cassette are arranged bidirectional for the selection of a transient transfected cell.

117. The method according to claim 110, wherein the first expression cassette and the second expression cassette are arranged bidirectional for the selection of a transient transfected cell.

118. The method according to claim 111, wherein the first expression cassette and the second expression cassette are arranged bidirectional for the selection of a transient transfected cell.

119. The method according to claim 109, wherein the expression plasmid further comprises a selection marker.

120. The method according to claim 118, wherein the expression plasmid further comprises a selection marker.

121. The method according to claim 117, wherein the expression plasmid further comprises a selection marker.

122. The method according to claim 118, wherein the expression plasmid further comprises a selection marker.

123. The method according to claim 109, wherein the expression cassettes and the selection marker are arranged unidirectional.

124. The method according to claim 119, wherein the expression cassettes and the selection marker are arranged unidirectional.

125. The method according to claim 109, wherein the expression cassettes are arranged in the sequence LC-HC-SM.

126. The method according to claim 119, wherein the expression cassettes are arranged in the sequence LC-HC-SM.

127. The method according to claim 123, wherein the expression cassettes are arranged in the sequence LC-HC-SM.

128. The method according to claim 109, wherein the mammalian cell is selected from CHO cell, HEK cell, BHK cell, NS0 cell, and SP2/0 cell.

129. The method according to claim 128, wherein the mammalian cell is a CHO cell for the selection of a stable transfected cell.

130. The method according to claim 128, wherein the mammalian cell is a CHO cell for the selection of a stable transfected cell.

131. The method according to claim 128, wherein the mammalian cell is a HEK cell for the selection of a transient transfected cell.

132. The method according to claim 128, wherein the mammalian cell is a HEK cell for the selection of a transient transfected cell.

133. A method for producing an antibody comprising the following steps: a) cultivating a mammalian cell comprising a first expression cassette comprising in 5 to 3 direction a hCMV promoter, a nucleic acid encoding an antibody light chain, a bGH polyA signal sequence, and a hGT terminator sequence, a second expression cassette comprising in 5 to 3 direction a hCMV promoter, a nucleic acid encoding an antibody heavy chain, a bGH polyA signal sequence, and a hGT terminator sequence, and b) recovering the antibody from the cell or the cultivation medium.

134. The method according to claim 133, wherein the nucleic acid encoding the antibody light chain and/or the nucleic acid encoding the antibody heavy chain comprises at least one intron.

135. The method according to claim 133, wherein the nucleic acid encoding the antibody light chain and/or the nucleic acid encoding the antibody heavy chain is cDNA.

136. The method according to claim 133, wherein the nucleic acid encoding the antibody light chain and/or the nucleic acid encoding the antibody heavy chain is cDNA.

137. The method according to claim 133, wherein the first expression cassette and the second expression cassette are arranged unidirectional for the stable production of an antibody.

138. The method according to claim 133, wherein the first expression cassette and the second expression cassette are arranged bidirectional for the transient production of an antibody.

139. The method according to claim 133, wherein the expression plasmid further comprises a selection marker.

140. The method according to claim 139, wherein the expression cassettes and the selection marker are arranged unidirectional.

141. The method according to claim 139, wherein the expression cassettes are arranged in the sequence LC-HC-SM.

142. The method according to claim 133, wherein the mammalian cell is selected from CHO cell, HEK cell, BHK cell, NS0 cell, and SP2/0 cell.

143. The method according to claim 142, wherein the mammalian cell is a CHO cell for the stable production of an antibody.

144. The method according to claim 142, wherein the mammalian cell is a HEK cell for the transient production of an antibody.

145. An expression vector comprising: a first expression cassette comprising in 5 to 3 direction a hCMV promoter, a nucleic acid encoding an antibody light chain, a bGH polyA signal sequence, and a hGT terminator sequence, and a second expression cassette comprising in 5 to 3 direction a hCMV promoter, a nucleic acid encoding an antibody heavy chain, a bGH polyA signal sequence, and a hGT terminator sequence.

146. The expression vector according to claim 145, wherein the nucleic acid encoding the antibody light chain and/or the nucleic acid encoding the antibody heavy chain comprises at least one intron.

147. The expression vector according to claim 145, wherein the nucleic acid encoding the antibody light chain and/or the nucleic acid encoding the antibody heavy chain is cDNA.

148. The expression vector according to claim 145, wherein the first expression cassette and the second expression cassette are arranged unidirectional for the selection of a stable transfected cell.

149. The expression vector according to claim 145, wherein the first expression cassette and the second expression cassette are arranged bidirectional for the selection of a transient transfected cell.

150. The expression vector according to claim 145, wherein the expression plasmid further comprises a selection marker.

151. The expression vector according to claim 145, wherein the expression cassettes and the selection marker are arranged unidirectional.

152. The expression vector according to claim 145, wherein the expression cassettes are arranged in the sequence LC-HC-SM.

153. A method for selecting a recombinant mammalian cell, comprising the steps of: a) transfecting a mammalian cell with an expression vector comprising a first expression cassette comprising in 5 to 3 direction a hEF1alpha promoter, a nucleic acid encoding an antibody light chain, and a bGH polyA signal sequence, a second expression cassette comprising in 5 to 3 direction a hEF1alpha promoter, a nucleic acid encoding an antibody heavy chain, a bGH polyA signal sequence, and thereby obtaining a multitude of recombinant mammalian cells, and b) selecting from the multitude of recombinant mammalian cells a (single) recombinant mammalian cell.

154. The method according to claim 153, wherein the nucleic acid encoding the antibody light chain and/or the nucleic acid encoding the antibody heavy chain comprises at least one intron.

155. The method according to claim 153, wherein the nucleic acid encoding the antibody light chain and/or the nucleic acid encoding the antibody heavy chain is cDNA.

156. The method according to claim 153, wherein the first expression cassette and the second expression cassette are arranged unidirectional for the selection of a stable transfected cell.

157. The method according to claim 153, wherein the first expression cassette and the second expression cassette are arranged bidirectional for the selection of a transient transfected cell.

158. The method according to claim 153, wherein the expression plasmid further comprises a selection marker.

159. The method according to claim 158, wherein the expression cassettes and the selection marker are arranged unidirectional.

160. The method according to claim 158, wherein the expression cassettes are arranged in the sequence LC-HC-SM.

161. The method according to claim 153, wherein the human elongation factor 1 alpha promoter comprises an Intron A.

162. The method according to claim 153, wherein the expression vector is free of any transcription terminator sequences.

163. The method according to claim 162, wherein the terminator sequence is the hGT sequence.

164. The method according to claim 162, wherein the mammalian cell is selected from CHO cell, HEK cell, BHK cell, NS0 cell, and SP2/0 cell.

165. The method according to claim 162, wherein the mammalian cell is a CHO cell for the selection of a stable transfected cell.

166. The method according to claim 162, wherein the mammalian cell is a HEK cell for the selection of a transient transfected cell.

167. A method for producing an antibody, comprising the steps of: a) cultivating a mammalian cell comprising a first expression cassette comprising in 5 to 3 direction a hEF1 alpha promoter, a nucleic acid encoding an antibody light chain, and a bGH polyA signal sequence, and a second expression cassette comprising in 5 to 3 direction a hEF1 alpha promoter, a nucleic acid encoding an antibody heavy chain, and a bGH polyA signal sequence, b) recovering the antibody from the cell or the cultivation medium.

168. The method according to claim 167, wherein the nucleic acid encoding the antibody light chain and/or the nucleic acid encoding the antibody heavy chain comprises at least one intron.

169. The method according to claim 167, wherein the nucleic acid encoding the antibody light chain and/or the nucleic acid encoding the antibody heavy chain is cDNA.

170. The method according to claim 167, wherein the first expression cassette and the second expression cassette are arranged unidirectional for the selection of a stable transfected cell.

171. The method according to claim 167, wherein the first expression cassette and the second expression cassette are arranged bidirectional for the selection of a transient transfected cell.

172. The method according to claim 167, wherein the expression plasmid further comprises a selection marker.

173. The method according to claim 172, wherein the expression cassettes and the selection marker are arranged unidirectional.

174. The method according to claim 172, wherein the expression cassettes are arranged in the sequence LC-HC-SM.

175. The method according to claim 167, wherein the human elongation factor 1 alpha promoter comprises an Intron A.

176. The method according to claim 167, wherein the expression vector is free of any transcription terminator sequences.

177. The method according to claim 176, wherein the terminator sequence is the hGT sequence.

178. The method according to claim 167, wherein the mammalian cell is selected from CHO cell, HEK cell, BHK cell, NS0 cell, and SP2/0 cell.

179. The method according to claim 178, wherein the mammalian cell is a CHO cell for the stable production of an antibody.

180. The method according to claim 178, wherein the mammalian cell is a HEK cell for the transient production of an antibody.

181. An expression vector, comprising: a first expression cassette comprising in 5 to 3 direction a hEF1 alpha promoter, a nucleic acid encoding an antibody light chain, and a bGH polyA signal sequence, and a second expression cassette comprising in 5 to 3 direction a hEF1 alpha promoter, a nucleic acid encoding an antibody heavy chain, and a bGH polyA signal sequence.

182. The expression vector according to claim 181, wherein the nucleic acid encoding the antibody light chain and/or the nucleic acid encoding the antibody heavy chain comprises at least one intron.

183. The expression vector according to claim 181, wherein the nucleic acid encoding the antibody light chain and/or the nucleic acid encoding the antibody heavy chain is cDNA.

184. The expression vector according to claim 181, wherein the first expression cassette and the second expression cassette are arranged unidirectional for the selection of a stable transfected cell.

185. The expression vector according to claim 181, wherein the first expression cassette and the second expression cassette are arranged bidirectional for the selection of a transient transfected cell.

186. The expression vector according to 181, wherein the expression plasmid further comprises a selection marker.

187. The expression vector according to claim 181, wherein the expression cassettes and the selection marker are arranged unidirectional.

188. The expression vector according to claim 181, wherein the expression cassettes are arranged in the sequence LC-HC-SM.

189. The expression vector according to claim 181, wherein the human elongation factor 1 alpha promoter comprises an Intron A.

190. The expression vector according to 181, wherein the expression vector is free of any transcription terminator sequences.

191. The expression vector according to claim 190, wherein the terminator sequence is the hGT sequence.

192. A method for producing a stable recombinant antibody in a mammalian cell, comprising the steps of: a) using a first expression cassette comprising in 5 to 3 direction a first promoter, a nucleic acid encoding an antibody light chain, a first polyA signal sequence, and optional a first transcription terminator sequence; b) using a second expression cassette comprising in 5 to 3 direction a second promoter, a nucleic acid encoding an antibody heavy chain, a second polyA signal sequence, and optional a second transcription terminator sequence; and c) using a third expression cassette comprising in 5 to 3 direction a third promoter, a nucleic acid conferring resistance to a selection agent, a third polyA signal sequence, and optional a third transcription terminator sequence, wherein the three expression cassettes are organized unidirectional and in the sequence first expression cassette-second expression cassette-third expression cassette.

193. The method according to claim 192, wherein the nucleic acid encoding the antibody light chain and/or the nucleic acid encoding the antibody heavy chain comprises at least one intron.

194. The method according to claim 192, wherein the nucleic acid encoding the antibody light chain and/or the nucleic acid encoding the antibody heavy chain is cDNA.

195. The method according to claim 192, wherein the first and the second promoter are the hCMV promoter, the first and second polyA signal sequence are the bGH polyA signal sequence, and the transcription termination sequence is present and is the hGT terminator sequence.

196. The method according to claim 192, wherein the first and the second promoter are the hEF1 alpha promoter, the first and second polyA signal sequence are the bGH polyA signal sequence, and the expression cassettes are free of a transcription terminator sequence.

197. The method according to claim 192, wherein the mammalian cell is selected from CHO cell, HEK cell, BHK cell, NS0 cell, and SP2/0 cell.

198. The method according to claim 192, wherein the mammalian cell is a CHO cell.

199. An expression vector, comprising: a) a first expression cassette comprising in 5 to 3 direction a first promoter, a nucleic acid encoding an antibody light chain, a first polyA signal sequence, and optional a first transcription terminator sequence; b) a second expression cassette comprising in 5 to 3 direction a second promoter, a nucleic acid encoding an antibody heavy chain, a second polyA signal sequence, and optional a second transcription terminator sequence; and c) a third expression cassette comprising in 5 to 3 direction a third promoter, a nucleic acid conferring resistance to a selection agent, a third polyA signal sequence, and optional a third transcription terminator sequence, whereby the three expression cassettes are organized unidirectional and in the sequence first expression cassette-second expression cassette-third expression cassette.

200. The expression vector according to claim 199, wherein the nucleic acid encoding the antibody light chain and/or the nucleic acid encoding the antibody heavy chain comprises at least one intron.

201. The expression vector according to claim 199, wherein the nucleic acid encoding the antibody light chain and/or the nucleic acid encoding the antibody heavy chain is cDNA.

202. The expression vector according to claim 199, wherein the first and the second promoter are the hCMV promoter, the first and second polyA signal sequence are the bGH polyA signal sequence, and the transcription termination sequence is present and is the hGT terminator sequence.

203. The expression vector according to claim 199, wherein the first and the second promoter are the hEF1 alpha promoter, the first and second polyA signal sequences are the bGH polyA signal sequence, and the expression cassettes are free of a transcription terminator sequence.

204. A method for the transient recombinant production of an antibody in a mammalian cell, comprising the steps of: a) using a first expression cassette comprising in 5 to 3 direction a first promoter, a nucleic acid encoding an antibody light chain, a first polyA signal sequence, and optional a first transcription terminator sequence; b) using a second expression cassette comprising in 5 to 3 direction a second promoter, a nucleic acid encoding an antibody heavy chain, a second polyA signal sequence, and optional a second transcription terminator sequence; and c) using a third expression cassette comprising in 5 to 3 direction a third promoter, a nucleic acid conferring resistance to a selection agent, a third polyA signal sequence, and optional a third transcription terminator sequence, whereby the expression cassettes are organized bidirectional whereby the first expression cassette and the second expression cassette are arranged in opposite direction.

205. The method according to claim 204, wherein the nucleic acid encoding the antibody light chain and/or the nucleic acid encoding the antibody heavy chain comprises at least one intron.

206. The method according to claim 204, wherein the nucleic acid encoding the antibody light chain and/or the nucleic acid encoding the antibody heavy chain is cDNA.

207. The method according to claim 204, wherein the first and the second promoter are the hCMV promoter, the first and second polyA signal sequence are the bGH polyA signal sequence, and the transcription termination sequence is present and is the hGT terminator sequence.

208. The method according to claim 204, wherein the first and the second promoter are the hEF1 alpha promoter, the first and second polyA signal sequence are the bGH polyA signal sequence, and the expression cassettes are free of a transcription terminator sequence.

209. The method according to claim 204, wherein the mammalian cell is selected from CHO cell, HEK cell, BHK cell, NS0 cell, and SP2/0 cell.

210. The method according to claim 204, wherein the mammalian cell is a HEK cell.

211. An expression vector, comprising: a) a first expression cassette comprising in 5 to 3 direction a first promoter, a nucleic acid encoding an antibody light chain, a first polyA signal sequence, and optional a first transcription terminator sequence; b) a second expression cassette comprising in 5 to 3 direction a second promoter, a nucleic acid encoding an antibody heavy chain, a second polyA signal sequence, and optional a second transcription terminator sequence, and c) a third expression cassette comprising in 5 to 3 direction a third promoter, a nucleic acid conferring resistance to a selection agent, a third polyA signal sequence, and optional a third transcription terminator sequence, whereby the expression cassettes are organized bidirectional whereby the first expression cassette and the second expression cassette are arranged in opposite direction.

212. The expression vector according claim 211, wherein the nucleic acid encoding the antibody light chain and/or the nucleic acid encoding the antibody heavy chain comprises at least one intron.

213. The expression vector according to claim 211, wherein the nucleic acid encoding the antibody light chain and/or the nucleic acid encoding the antibody heavy chain is cDNA.

214. The expression vector according to claim 211, wherein the second promoter are the hCMV promoter, the first and second polyA signal sequence are the bGH polyA signal sequence, and the transcription termination sequence is present and is the hGT terminator sequence.

215. The expression vector according to claim 211, wherein the first and the second promoter are the hEF1 alpha promoter, the first and second polyA signal sequences are the bGH polyA signal sequence, and the expression cassettes are free of a transcription terminator sequence.

216. An expression plasmid comprising in 5 to 3 direction a promoter sequence, a nucleic acid encoding an antibody heavy chain or an antibody light chain, an IRES element, a nucleic acid sequence encoding a selection marker, and a polyA signal sequence, whereby the IRES element is the EMCV-IRES element.

217. A method of using an expression cassette said method comprising: in 5 to 3 direction using a promoter sequence, a nucleic acid encoding an antibody heavy chain or an antibody light chain, an IRES element, a nucleic acid sequence encoding a selection marker, and a polyA signal sequence for the selection of antibody producing cells, whereby the IRES element is the EMCV-IRES element.

218. A method for the selection of eukaryotic cell expressing an antibody, comprising the steps of: a) cultivating a eukaryotic cell comprising i) an expression plasmid according to claim 107 and ii) a nucleic acid encoding the respective other antibody chain not encoded by the expression plasmid according to claim 107; and b) selecting a cell expressing the detectable polypeptide.

219. An expression plasmid comprising in 5 to 3 direction: a promoter sequence, a nucleic acid encoding an antibody light chain, an IRES element, a nucleic acid sequence encoding an antibody heavy chain, and a polyA signal sequence, whereby the IRES element is the EV71-IRES element.

220. A method of using an expression plasmid said method comprising: in 5 to 3 direction using a promoter sequence, a nucleic acid encoding an antibody light chain, an IRES element, a nucleic acid sequence encoding an antibody heavy chain, and a polyA signal sequence, for the expression of an antibody, whereby the IRES element is the EV71-IRES element.

221. A method for the selection of eukaryotic cell expressing an antibody, comprising the steps of: a) cultivating a eukaryotic cell comprising i) an expression vector according to claim 110 and ii) a nucleic acid encoding the respective other antibody chain not encoded by the expression vector according to claim 110; and b) selecting a cell expressing the detectable polypeptide.

222. The method according to claim 109, wherein the expression vector comprises: a) a first expression cassette comprising in 5 to 3 direction a promoter, a nucleic acid encoding a first antibody light chain, a polyA signal sequence, and optionally a terminator sequence; b) a second expression cassette comprising in 5 to 3 direction a promoter, a nucleic acid encoding a second antibody light chain, a polyA signal sequence, and optionally a terminator sequence; c) a third expression cassette comprising in 5 to 3 direction a promoter, a nucleic acid encoding a first antibody heavy chain, a polyA signal sequence, and optionally a terminator sequence; and d) a fourth expression cassette comprising in 5 to 3 direction a promoter, a nucleic acid encoding a second antibody heavy chain, a polyA signal sequence, and optionally a terminator sequence.

223. The method according to claim 109, wherein the expression vector comprises: a) a first expression cassette comprising in 5 to 3 direction a promoter, a nucleic acid encoding an antibody light chain, a polyA signal sequence, and optionally a terminator sequence; b) a second expression cassette comprising in 5 to 3 direction a promoter, a nucleic acid encoding a first antibody heavy chain, a polyA signal sequence, and optionally a terminator sequence; and c) a third expression cassette comprising in 5 to 3 direction a promoter, a nucleic acid encoding a second antibody heavy chain, a polyA signal sequence, and optionally a terminator sequence, wherein the antibody light chain is a common light chain for both antibody heavy chains.

224. The method according to claim 222, wherein the expression vector is encoding a bispecific antibody.

225. The method according to claim 222, wherein the bispecific antibody has a first binding specificity or binding site that specifically binds to a first antigen or a first epitope on an antigen and the bispecific antibody has a second binding specificity or binding site that specifically binds to a second antigen or second epitope on the antigen.

226. The method according to claim 222, wherein said expression vector comprises: a) an antibody light chain expression cassette; b) a first antibody heavy chain expression cassette; c) a second antibody heavy chain expression cassette; and d) a selection marker expression cassette.

227. The method according to claim 222, wherein said expression vector comprises: a) an antibody light chain expression cassette; b) a first antibody heavy chain expression cassette; c) a second antibody heavy chain expression cassette; and d) a selection marker expression cassette.

228. The method according to claim 226, wherein the expression vector comprises a) a first antibody light chain expression cassette; b) a second antibody light chain expression cassette; c) a first antibody heavy chain expression cassette; d) a second antibody heavy chain expression cassette; and e) a selection marker expression cassette.

229. The method according to claim 227, wherein the expression vector comprises a) a first antibody light chain expression cassette; b) a second antibody light chain expression cassette; c) a first antibody heavy chain expression cassette; d) a second antibody heavy chain expression cassette; and e) a selection marker expression cassette.

230. The method according to claim 227, wherein the antibody heavy chain expression cassettes encodes an antibody heavy chain comprising a hole mutation.

231. The method according to claim 228, wherein the antibody heavy chain expression cassettes encodes an antibody heavy chain comprising a hole mutation.

232. The method according to claim 229, wherein one of the antibody heavy chain expression cassettes encodes an antibody heavy chain comprising a knob mutation.

233. The method according to claim 230, wherein one of the antibody heavy chain expression cassettes encodes an antibody heavy chain comprising a knob mutation.

234. The method according to claim 229, wherein one of the antibody light chain expression cassettes encodes an antibody light chain variant comprising an antibody light chain variable domain and an antibody heavy chain CH1 domain as constant domain and/or one of the antibody light chain expression cassettes encodes an antibody light chain comprising an antibody light chain variable domain and an antibody light chain CL domain as constant domain.

235. The method according to claim 229, wherein one of the antibody heavy chain expression cassettes encodes an antibody heavy chain variant comprising as first constant domain an antibody light chain constant domain (CL), and/or one of the antibody heavy chain expression cassettes encodes an antibody heavy chain comprising as first constant domain an antibody heavy chain CH1 domain.

Description

FIGURES

[0616] FIG. 1 Productivity of stable clones generated with the vectors p5068, px6001, px6008 and px6007. Shown is the average productivity of the best 15 clones obtained with each vector in batch analysis of a total of three independent transfections.

[0617] FIG. 2 Productivity of stable clones generated with the vectors px6051, px6062, px6052, and px6063. Shown is the average productivity of the best 18 clones each vector in batch analysis of a total of three independent transfections.

[0618] FIG. 3 Productivity of vectors p5068, px6051, px6052 and px6053 in transient transfections using the 96we11 shuttle system from Amaxa. Shown is the average productivity of eight independent transfections each vector on day 4 after transfection measured by ELISA.

[0619] FIG. 4 Productivity of different stable pools generated with the vectors p5068, px6051, px6052, and px6053 in batch analysis. Shown is the average productivity of three pools each vector on day 7.

[0620] FIG. 5 Productivity of stable pools generated with the vectors p5069 and px6010C. Shown is the average productivity of two (px5069) respectively three (px6010C) different pools each vector in batch analysis on day 7.

[0621] FIG. 6 Stability of gene expression in stable pools generated with the vectors p5069 and px6010C. Shown is the average productivity of two different pools each vector in batch analysis on day 7 at passage 0 (set to 100%, black column) and at generation 30 with (white column) and without selection (patterned column) pressure (G418).

[0622] FIG. 7 Productivity of the best 15 clones generated by the vectors p5069 and px6010C. Average productivity of the best 15 clones each vector in batch analysis of a total of two independent transfections.

[0623] FIG. 8 Schematic overview on the vector design of vector px6011C mediating the IRES mediated expression of the GFP-PEST-NEO fusion protein. The GFP-PEST-Neo fusion protein is linked by the EMCV-IRES to the heavy chain of the antibody. The coding sequences of the heavy chain of the antibody and of the fusion protein are transcribed by the short human CMV promoter in one mRNA. The translation of this mRNA leads to the heavy chain of the antibody and to the GFP-PEST-NEO fusion protein.

[0624] FIG. 9 Productivity of different stable pools generated by the vectors p5069, px6011C and px6010C in batch analysis. Shown is the average productivity of two different pools each vector on day 7.

[0625] FIG. 10 Productivity of the best 15 clones generated by the vectors p5069, px6010C (vector expressing the selection marker Neomycin by the EMCV-IRES element that is linked to the heavy chain of the antibody) and px6011C (vector expressing the fusion protein of GFP-PEST-Neomycin by the EMCV-IRES element that is linked to the heavy chain of the antibody). (A) Productivity distribution of the best 15 clones each vector in batch analysis of a total of two independent transfections. (B) Average productivity of the best 15 clones each vector in batch analysis of a total of two independent transfections.

[0626] FIG. 11 Shown is the dependence of the GFP expressing level/fluorescence intensity and productivity in batch analysis for 11 clones generated with vector px6011C. Clones were picked randomly in the 24 well screening format, then expanded and finally analyzed in batch analysis. The geometric mean (GM) of the GFP fluorescence intensity and the percentage of GFP-positive cells of each clone were determined by FACS. (A) Dependence of GFP fluorescence intensity and productivity in batch analysis for 11 single clones. (B) Dependence of the percentage of GFP-positive cells each clone and productivity in batch analysis for 11 single clones.

[0627] FIG. 12 Productivity of stable pools with different GFP fluorescence intensities in batch analysis. Cells with different GFP expressing levels/fluorescence intensities (low (1), medium (2) and high (3)) were sorted by FACS. Pools were expanded and productivity of the pools was determined in batch analysis on day 7.

[0628] FIG. 13 Plasmid map of px6007.

[0629] FIG. 14 Plasmid map of px6053.

[0630] FIG. 15 Plasmid map of px6062.

EXAMPLES

[0631] Expression vector p5068 and p5069

[0632] Expression plasmids p5068 and p5069 comprise expression cassettes for the expression of an anti-P-selectin antibody (genomically organized expression cassette with retained exon-intron organization) as reported in WO 2005/100402.

[0633] The anti-P-selectin HuMab light and heavy chain encoding genes were separately assembled in mammalian cell expression vectors.

[0634] Thereby the gene segments encoding the anti-P-selectin HuMab light chain variable region (VL) and the human x-light chain constant region (CL) were joined as were gene segments for the anti-P-selectin HuMab heavy chain variable region

[0635] (VH) and the human yl-heavy chain constant region or the human y4-heavy chain constant region (CH1-Hinge-CH2-CH3).

[0636] General information regarding the nucleotide sequences of human light and heavy chains from which the codon usage can be deduced is given in: Kabat, E. A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication No 91-3242.

[0637] The transcription unit of the anti-P-selectin HuMab x-light chain is composed of the following elements: [0638] the immediate early enhancer and promoter from the human cytomegalovirus (hCMV), [0639] a synthetic 5-UT including a Kozak sequence, [0640] a murine immunoglobulin heavy chain signal sequence including the signal sequence intron, [0641] the cloned anti-P-selectin HuMab variable light chain cDNA arranged with a unique BsmI restriction site at the 5 end and a splice donor site and a unique NotI restriction site at the 3 end, [0642] the genomic human ic-gene constant region, including the intron 2 mouse Ig- enhancer (Picard, D., and Schaffner, W. Nature 307 (1984) 80-82), and [0643] the human immunoglobulin x-polyadenylation (poly A) signal sequence.

[0644] The transcription unit of the anti-P-selectin HuMab l-heavy chain is composed of the following elements: [0645] the immediate early enhancer and promoter from the human cytomegalovirus (hCMV), [0646] a synthetic 5-UT including a Kozak sequence, [0647] a modified murine immunoglobulin heavy chain signal sequence including the signal sequence intron, [0648] the cloned anti-P-selectin HuMab variable heavy chain cDNA arranged with a unique BsmI restriction site at the 5 and a splice donor site and a unique NotI restriction site at the 3 end, [0649] the genomic human l -heavy gene constant region, including the mouse Ig -enhancer (Neuberger, M. S., EMBO J. 2 (1983) 1373-1378), and [0650] the human l-immunoglobulin polyadenylation (polyA) signal sequence.

[0651] Beside the anti-P-selectin HuMab -light chain or l-heavy chain expression cassette these plasmids contain [0652] a hygromycin resistance gene, [0653] an origin of replication, oriP, of Epstein-Barr virus (EBV), [0654] an origin of replication from the vector pUC18 which allows replication of this plasmid in E. coli, and [0655] a -lactamase gene which confers ampicillin resistance in E. coli.

[0656] Recombinant DNA Techniques

[0657] Cloning was performed using standard cloning techniques as described in Sambrook et al., 1999 (supra). All molecular biological reagents were commercially available (if not indicated otherwise) and were used according to the manufacturer's instructions.

[0658] Nucleic Acid Synthesis

[0659] DNA of the different genetic elements was synthesized by Geneart AG,

[0660] Regensburg.

[0661] Nucleic Acid Sequence Determination

[0662] DNA sequences were determined by double strand sequencing performed at SequiServe (SequiServe GmbH, Germany).

[0663] DNA and Protein Sequence Analysis and Sequence Data Management

[0664] The Vector NTI Advance suite version 9.0 was used for sequence creation, mapping, analysis, annotation, and illustration.

[0665] Cell Culture Techniques

[0666] CHO-K1 cells were grown in CD-CHO medium (Invitrogen Corp., Gibco, Cat. No. 10743-011) supplemented with 1 HT supplement (Invitrogen Corp., Gibco, Cat. No. 11067-030).

[0667] For the selection of stably transfected CHO-K1 pools/cells lines 400 to 800 g/ml G418 or 200 to 400 g/ml Hygromycin was added (Roche Diagnostics GmbH, Roche Applied Sciences, Germany, Cat. No.: 843555).

[0668] All cell lines were maintained in humidified incubators at 37 C. with 5% CO.sub.2 under constant agitation at 120 to 140 rpm/min. Every 3 to 4 days the cells were split into fresh medium. Density and viability of the cultures was determined using the Casey TT or Cedex Hires cell counter (Roche innovates AG, Bielefeld). Transfection of cells was performed by the Amaxa nucleofection technology (Lonza GmbH, Germany).

[0669] Furthermore standard cell culture techniques were applied as described e.g. in Bonifacino, J. S., et al., (eds.), Current Protocols in Cell Biology, John Wiley and Sons, Inc. (2000).

[0670] Cell Counting and Determination of Cell Viability

[0671] a) Electric field cell counting system (CASY)

[0672] The CASY Technology Cell Counter, Model TT (Roche Innovatis AG, Bielefeld) uses electric current for cell counting. The Pulse Area Analysis was used to get information from signals created when a cell passes through the measuring pore in a low voltage field. The structural integrity of the cell membrane is a degree for cell viability. Dyes such as trypan blue are therefore not needed for determination of viability.

[0673] b) Automated trypan blue exclusion method (Cedex)

[0674] A Cedex HiRes system (Roche Innovatis AG, Bielefeld) was used to determine cell viabilities during pool selections and for automated cell counting.

[0675] Trypan blue is a dye that cannot enter cells through intact cell membranes. Only those cells are stained, and marked dead, which have a damaged cell membrane. The staining process, cell counting and graphical analysis of the results were performed automatically by the Cedex system by digital image recognition. Other measurement parameters are cell size, morphology and aggregation rate. With the multi sampler, up to 20 samples were measured consecutively.

[0676] Plasmid Preparation and Quality Check for Accurate Comparison of Plasmids in Transfections

[0677] Transfection efficacy and therefore productivity is strongly influenced by several factors such as DNA amount and quality. To ensure equal starting conditions for each vector the DNA amount and quality of all vectors were intensively checked before transfection. [0678] Simultaneously preparation of expression vectors

[0679] All vectors were simultaneously prepared by the High Speed Maxi plasmid isolation Kit (Qiagen GMBH, Hilden) according to manufactures' instructions. [0680] Phenol/chloroform purification and ethanol precipitation

[0681] All vectors were simultaneously purified by a phenol/chloroform purification. 500 g each linearized plasmid DNA was mixed with 200 l Tris-buffered 50% (v/v) phenol, 48% (v/v) chloroform, 2% (v/v) isoamyl alcohol solution and centrifuged for 1 min. at 13,000 rpm. The upper aqueous phase was then transferred into a new tube and mixed with 200 l 96% (v/v) chloroform, 4% (v/v) isoamyl alcohol and centrifuged for 1 min. at 13;000 rpm. The upper phase was again transferred into a new tube and mixed with 1/10 (total volume) 3 M sodium acetate (pH 5.2) and 2.5 times (total volume) 100% ethanol. After mixing and incubating the reaction for 5 min. at room temperature, the mixture was centrifuged for 5 min. at 13,000 rpm in order to pellet the DNA. The supernatant was discarded and the pellet was washed with 900 l 70% (v/v) ethanol and incubated for 5 min. at room temperature. After a final centrifugation step at maximum speed for 5 min., the supernatants were discarded and the pellets were dried and resuspended with sterile H.sub.2O. [0682] DNA determination

[0683] The DNA amount of each vector was determined using the BioPhotometer (Eppendorf; Hamburg). DNA measurement was always performed in triplicates by using a 1:20 dilution in Tris pH 8.0). [0684] Agarose gel

[0685] DNA quality of each plasmid was checked on a 0.8% agarose gel. DNA degradation, vector conformations and DNA concentrations were determined. Vectors showing comparable quantities and qualities (no DNA degradation, similar supercoiled (ccc) forms, similar DNA amounts on gel) were used for transient and stable transfections.

[0686] Transient Transfections

[0687] All vectors were transfected in CHO-K1 cells by the Amaxa 96 well shuttle system (Lonza GmbH, Germany) according to manufactures' instructions. Each vector was transfected in 8 replicates. DNA amounts of transfected vector were normalized to equal molar amounts/copy numbers according to 1 g of the reference expression plasmid (p5068 or p5069). To determine productivity cell free cell culture supernatant was analyzed for IgG titer on day 4 to 7 after transfection by a one-step universal ELISA (Dianova).

[0688] Amaxa 96 well shuttle system:

[0689] CHO-K1 cells growing in spinner flasks were pelleted by centrifugation at 850 rpm for 5 min. and resuspended in culture medium. Circular plasmids were plated out in 96-well nucleofection plates at equimolar concentrations according to 1 g of the reference expression vector p5068 or p5069. Cells were then added into the plates at a concentration of 410.sup.5 cells per well. The transfection was carried out by the Amaxa program DN-137. Cells were incubated for 10 min. after transfection and then transferred into 96 well flat-bottom incubation plates containing 200 pl culture medium. Cells were then statically cultivated. On day 4 to 6 after transfection IgG levels were determined using the one-step universal ELISA.

[0690] Stable Transfections and Generation of Recombinant CHO Cell Lines

[0691] Stable transfections were performed by the nucleofection technology (Amaxa Biosystems, Lonza cologne AG) according to manufactures' instructions. Before transfection plasmids were linearized by the restriction enzyme SgrA I. Each plasmid was transfected in duplicates or triplicates. 510.sup.6 cells and 1.2 pmol linearized plasmid were used per single transfection. (Nucleofector Kit T, Amaxa program A33).

[0692] For transfection cells were resuspended in the Nucleofector solution T and aliquoted into 2 ml tubes. After the addition of the plasmid, the transfection was carried out by applying the pulse. Cells were then transferred into T25 tissue culture flasks containing pre-warmed 4 ml fresh medium and 4 ml conditioned medium. Selective pressure was applied 24 hours post-transfection by adding 250 g/ml Hygromycin B.

[0693] Generation of Stable Pools

[0694] Vectors were transfected into CHO-K1 cells by Amaxa nucleofection technology and stable pools were selected using Hygromycin B or G418 as selection agent. Each transfection was performed in triplicates. For generation of stable pools, all plasmids were uniformly linearized by restriction digestion with SgrA I. The Nucleofector Kit T by Amaxa was used for carrying out stable transfections and each plasmid was transfected in triplicates.

[0695] Stable pools were established as follows: 510.sup.6 cells and 1.2 pmol linearized plasmid were used for each transfection. Cells were resuspended in Solution T and aliquoted into 2 ml tubes. After the addition of the plasmid, the transfection was carried out by applying the pulse (Amaxa program A33). The transfected cell pools were statically cultivated in T25 tissue culture flasks containing pre-warmed 4 ml fresh medium and 4 ml conditioned medium.

[0696] After 24 h post-transfection selection pressure was applied: Cells were centrifuged for 5 min. at 800 rpm, resuspended in 3 ml culture medium containing 300 g/ml Hygromycin B. Cells were transferred into flat-bottom 6-well plates 3 days post-transfection. Cells were then cultivated for two weeks till cell viabilities dropped to a minimum and rose again over 99%. Cell numbers and viabilities were constantly determined with a Cedex HiRes system (Innovatis, Bielefeld). During cultivation, cell debris was removed by centrifugation and cells were always resuspended in 3 ml fresh medium.

[0697] Generation of Stable Clones using the Caliper Robotic System

[0698] Vectors were transfected into CHO-K1 cells as described above. 48 hours after transfection selection pressure was applied (Hygromycin B or G418) and cells were seeded onto 384 well flat-bottom plates at a concentration of 350 to 700 cells per well using an automated high-throughput clone isolation system (Sciclone ALH 3000 workstation, Caliper Life Sciences GmbH, Mainz).

[0699] After 10 to 14 days the 384 well plates were screened for IgG levels using an ELISA based ultrahigh-throughput screening (ELSIA uHTS). From a primary screening the best producing clones were chosen and transferred into flat-bottom 96 well plates. After 3 to 6 days cells were screened for IgG levels in a second round. The best producing clones again were chosen and manually transferred into flat-bottom 24 well plates. After a further ELISA based screening step the best clones were chosen and transferred into flat-bottom 6 well plates. IgG levels in the 6-well plates were determined by ProtA measurement to identify the final best clones for batch culture in shaken 6 well plates.

[0700] Batch Analysis of Pools/Single Clones

[0701] In order to detect differences in productivity and stability, cell numbers of the clones/pools were counted using a Casey cell counter and uniformly seeded into flat-bottom 6 well plates at a concentration of 310.sup.5 cells/ml and a total volume of 3.0 ml. All batch cultures were cultivated for 12 days and cell culture supernatants were screened for human IgG levels at day 4, 7, 9, 11 or 12.

[0702] IgG Quantification

[0703] The IgG titer in transient experiments and in the screening formats (384 well to 24-well) were determined by using the one-step universal ELISA. Productivity of stable pools and stable single clones in batch experiments were determined by Protein A HPLC.

[0704] One-Step Universal ELISA

[0705] A one-step universal ELISA (Dianova) was used to determine human IgG levels from cell culture supernatants. A standard curve was prepared using serial dilutions of an anti-P-Selectin antibody (F. Hoffmann-La Roche AG, Basle, Switzerland) with a range of 0.3125-20 ng/ml using dilution buffer (PBS+5% (w/v) RPLA1). 95 pl antibody-mix containing 0.5 g/ml biotinylated F(ab).sub.2-anti-human Fc antibody (Jackson laboratories) and 0.1 g/ml peroxidase conjugated F(ab').sub.2-anti-human Fc antibody (Jackson laboratories; Suffolk) was added to streptavidin-coated 96-well MTP (StreptaWell, Roche Diagnostics GmbH). 5 l of 1:20.000 diluted cell culture supernatant was added to the plates and incubated for 1 hour. Antibody coated plates were washed three times with 200 1 washing buffer (PBS+0.05% (v/v) Tween.sup.20). 100 l ABTS (Roche Diagnostics GmbH, Mannheim, Germany) was added to the plates and the absorbance was measured at 405 nm with a reference wavelength of 492 nm.

[0706] ProtA-measurement

[0707] The IgG titer of batch analysis were determined by Protein A using a HPLC based chromatography in combination with the one-step universal ELISA.

[0708] FACS

[0709] Fluorescence-activated cell sorting was used to determine transfection efficiencies (based on GFP expressing cells) or GFP expression levels of stably or transiently transfected cells. In general 510.sup.6 cells each clone or pool were measured using a FACSCalibur Flow Cytometer (BD Biosciences, San Diego, Calif.). Forward and sideward scatter data were used to determine cell size, viability and cell morphology.