Important Topics in the Expression of Recombinant Antibodies from CHO Cells

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1 Cell Line Development and Engineering Workshop, Prague, March 2008 Important Topics in the Expression of Recombinant Antibodies from CHO Cells Robert Young/Lonza Biologics/Mar 2008

2 Disclaimer Certain matters discussed in this presentation may constitute forward-looking statements. These statements are based on current expectations and estimates of Lonza Group Ltd, although Lonza Group Ltd can give no assurance that these expectations and estimates will be achieved. The actual results may differ materially in the future from the forward-looking statements included in this presentation due to various factors. Furthermore, Lonza Group Ltd has no obligation to update the statements contained in this presentation. Note: All slides are incomplete without verbal comments. slide 2

3 Talk Outline Introduction Transcription and Expression Vector Technology Transcription: Lonza s Experience Enhancing translation Enhancing secretion Antibody isotype and expression levels Summary slide 3

4 Introduction Achieving high antibody concentrations is the combination of many factors including: Expression vector technology. Cell line. Media and feeds. Process. In order to improve the concentrations of recombinant protein achievable in a cell culture process, all aspects of the production process require optimisation. New technologies are emerging that can be applied to recombinant protein production. Focus on the application of new technologies to a commercially relevant expression platform. slide 4

5 The Question How can the steps involved in the central dogma of protein expression be manipulated to improve production of recombinant antibodies? From Gene to Protein Transcription Translation Secretion slide 5

6 Transcription and Expression Vector Technology

7 Key Features of mammalian expression Vectors (Ludwig 2006 Bioproc Int Supp. 4:14-23) slide 7

8 The importance of the position effect The level of transcription depends on the site of transgene integration Integration may occur in open chromatin or in a region of condensed inactive chromatin Most genomic sites repress transgene expression (in humans, 60 % of sites are silent) Extensive screening of clones required to isolate rare events of integration in active DNA Sequence elements have been used to reduce influence of the position effect For example; Insulators, MAR/SAR and STAR etc slide 8

9 Other approaches to improving transcription New or enhanced selectable markers (e.g. stringency) Stronger promoters (e.g. murine CMV vs human CMV) Other promoters/transcription units (e.g., CHEF1) Different vector approaches (e.g. retroviral vectors, artificial chromosomes, etc) slide 9

10 Insulator elements (1) (West and Fraser 2005 Hum Mol Genet. 14:R101-R111.) Insulators can be defined as: Sequence elements that possess a common ability to protect genes from inappropriate signals emanating from their surrounding environment (phenotypic definition) 5 HS4 element from chicken β-globin locus has blocking and barrier function: Prevents the action of gene enhancers when placed between enhancer and promoter sequence (blocking) Halt the progression of chromosome silencing, hence the formation of heterochromatin (barrier) Footprint IV sequence binds USF protein leading to recruitment of enzymes responsible for histone H3/H4 protein acetylation and H3K4 methylation Prevents encroachment of H3K9 methylation and chromatin condensation slide 10

11 Insulator elements (2) Usage Recillas-Targa 2002 Proc Natl Acad Sci (USA) 99: Flanking a transgene with two copies of an insulator can shield it from chromosomal position effect following stable integration The majority of chromosomal loci are transcriptional silent in mature mammalian tissues so consequently position effect manifests as unwanted transcriptional silencing slide 11

12 Stabilising and anti-repressor element (STAR) (Kwaks et al Nat. Biotech. 21, ) STAR elements were isolated using a genetic screen STAR element properties Transcription blocker Increase expression from the hcmv promoter Increase the number of colonies that expressed the transgene Stable expression of SEAP over 60 generations STAR-Select TM leads to 6x higher MAb yields and pg/cell/day with picking only 24 colonies (before process optimization) Stringency increased by linking transgene to zeomycin via an IRES slide 12

13 Expression Augmenting Sequence Element (EASE) vectors (Amgen) (Aldrich et al Biotechnol Prog. 19: ) Isolated from CHO cells proximal to a unique integration site of a recombinant TNF-Fc fusion protein (3.6 kb) One EASE element per transcription unit on separate light (neo) and heavy chain (dhfr) expression vectors were used. In each case an IRES linked the Ig gene to the relevant selectable marker Not a promoter/enhancer/mar/sar/lcr element and has no ORF so does not encode a transactivator Cloning of CS-9 (CHO DXB-11 serum-free suspension adapted variant) transfectants in 50 nm MTX-containing media EASE clones produced pcd (85-95% viability) and were stable over 20 generations Non-EASE clones produced pcd (28-41% viability). Conclusion was that EASE-vectors allowed for greater co-amplification of the antibody heavy and light chains with increased concentrations of MTX in the growth medium. Viability and stability were both enhanced. Mechanism of action is unknown, but it might be to facilitate integration into the host genome slide 13

14 Matrix Attachment Regions (MARs) AT-rich DNA elements that bind to the nuclear matrix. Not all AT rich regions are MARs. Not all MAR sub-fragments that bind nuclear matrix have the ability to enhance and insulate transgene expression (Phi-Van and Strätling 1996 Biochemistry 35, ) Classical MARs are located near the boundaries of active chromatin domains and may map with loop anchorage points (5 boundary of chicken lysozyme locus; Phi-Van and Strätling, 1988 EMBO J. 7:655-64) Often contain topoisomerase consensus sequences Their action in functional transgenic assays define them as classical insulators MARs are thought to be involved in creating high-order structuring of chromatin in topologically independent loop domains (open chromatin) Not much is known about how MARs work at the molecular level and why they can have beneficial effects on transgene expression They are relatively small (=<3 kb) and can be incorporated into vectors easily slide 14

15 CHEF1 Vectors (ICOS) (Running Deer et al Biotechnol Prog. 20, 880-9) 12Kb 5 and 4 kb 3 flanking sequences of a highly expressed CHO gene (CHEF1α) with EF-1α promoter Average pooled transfectant productivity with a Fc Fusion (DG44) was 6 to 35- fold higher than pcdna3 or pef1/v5 (human E1Fα promoter). Top clone produced ~130 mg/l CHEF1-driven expression of SEAP or CCR4 was 13 to 280-fold and 10-fold greater, respectively, than pcdna3 or pef1/v5 in non-cho lines MTX not required for stability slide 15

16 Stringency of selection NPT engineering (Sautter et al Biotech Bioeng. 89, 530-8) Incorporated into the commercial BI-HEX expression system (Boehringer-Ingelheim) Based on impairment of the activity of the selectable marker enzyme neomycin-phosphotransferase (NPT) The ratio of high producing cells to total was higher with the mutant NPT marker compared to the wild type With this system, higher levels of mutant NPT mrna, higher specific productivities and titres were achieved. Elevated productivities were ascribed to higher gene copy numbers, integration into sites of high transcriptional activity or a combination of both slide 16

17 Transcription: Lonza s Experience

18 Typical GS Vector for Antibody Expression Gene of interest driven by strong promoter hcmv-mie GS is inhibited by methionine sulphoximine GS cdna driven by weak promoter SV40E Increase selection stringency Weak promoter on GS gene - selects for rare integration into transcriptionally efficient sites in genome hcmv-mie promoter SV40 intron + Poly-A Light Chain Gene Intron A 5'UTR GS cdna 5'UTR SV40E (and SV40 ori) GS Vector bp SV40 Poly-A hcmv-mie promoter 5'UTR beta-lactamase Intron A Heavy Chain Gene SV40 Poly-A pee6 ori 5'UTR slide 18

19 Quantification of mrna levels As the site of integration into the genome is likely to have a major effect on transcription (position effect), a panel of cell lines expressing a model antibody with a variety of productivities was evaluated. Relative mrna levels of the heavy and light chain were determined by quantitative RT-PCR. Higher levels of mrna were associated with higher product concentrations. Relative Heavy Chain 1 0 Heavy Chain Relative Light Chain 1 0 Light Chain Col 1 vs Col 2 Plot 1 Regr Antibody concentration (mg/l) Antibody concentration (mg/l) slide 19

20 Alternative to the hcmv-mie Promoter The murine CMV-MIE (mcmv-mie) promoter has been reported to be stronger than the human alternative (hcmv-mie) (Addison et al., 1997 J.Gen.Vir. 78: , Rotodara et al., 1996 Gene 168: 195-8) To test this, GS expression vectors were constructed to enable expression of GFP using either the hcmv-mie promoter plus the hcmv-mie Intron A (as present in the GS vectors) or the mcmv- MIE promoter. Transfect CHO-K1 cells, plate into T175 flasks. Select with MSX for 26 days. Analyse pool of transfectants for fluorescence at 510 nm (excitation 488 nm) with Coulter Elite flow cytometer. Record mean channel number for the highest 10% of the viable cell population. slide 20

21 Alternative to the hcmv-mie Promoter Use of the mcmv-mie promoter resulted in increased mean fluorescence indicating increased protein expression is possible using this promoter Mean Channel Number Control cells hcmv + hcmv intron mcmv slide 21

22 Alternative Promoters Do the observed increases in GFP expression levels predict increased levels of a more complex secreted protein such as an antibody? Evaluated GS vectors encoding a model antibody utilising the hcmv-mie promoter and human Intron A. the mcmv-mie promoter. the mcmv-mie promoter and human Intron A. slide 22

23 Evaluating Vector Improvements Method for evaluating vector improvements. Transfect host cells with vector 3-4 weeks 100 transfectants 100 transfectants 2 weeks 100 data points Identify single colonies per well Transfer to 24 well plate Productivity assessment (quantitative) Compare against control slide 23

24 Antibody Expression using the mcmv-mie Promoter Use of the mcmv-mie promoter plus hcmv-mie Intron A resulted in equivalent mean antibody concentrations to the hcmv-mie promoter plus hcmv-mie Intron A. Use of mcmv-mie promoter alone resulted in reduced mean antibody concentrations. Antibody concentration (mg/l) * 0 mcmv mcmv + hcmv intron hcmv + hcmv intron * P < 0.05 vs hcmv/intron slide 24

25 Lonza s experience of MARs in the GS Expression System (1) MAR element used: 3 kb chicken lysozyme MAR element (Phi-Van et al Mol Cell Biol. 10, ). MAR element placed in 3 different locations within expression vector. 5 Mid 3 slide 25

26 Lonza s experience of MARs in the GS Expression System (2) slide 26

27 Why MARs weren t successful in the GS Expression System? Possible answers GS system biases integration into sites of highest transcriptional activity, hence masking the benefit of MAR elements The 5 chicken lysozyme MAR may not necessarily be the best choice The human β-globin MAR was found to be better in a recent screen of MARs. Used to simplify DHFR-based amplification/cell line selection (Kim et al J Biotechnol. 107, ) Latterly reported that the human β-interferon SAR (scaffold attachment region) was even more effective when 2 copies were used in the 5 and 3 flanking regions (Kim et al :933-37) Selexsis have better understanding of MAR and how to use them and can get CHO cell lines with Qp of up to 110 pcd (up to 65pcd from GS-CHO) slide 27

28 Conclusions High levels of transcription are important for maximal antibody productivity. Increasing levels of transcription is important to move from low to high producing cell lines. Alternative promoters can result in increased protein expression. However, this effect was not observed when the stronger promoter was used to express a complex secreted protein. What steps downstream of transcription can be manipulated to increase productivity? Can optimisation of these steps increase the amount of protein expressed from highly productive cell lines further. slide 28

29 Enhancing Translation

30 Gene Optimisation Gene optimisation aims to improve productivity of a given protein by altering the gene sequence without altering the protein it encodes. Codon optimisation Use of optimal codons for the desired host cell. Optimisation of RNA structure e.g. removing cryptic splice sites and poly A signals, optimising GC content, decreasing undesirable mrna folding. slide 30

31 Evaluation of Gene Optimisation Genes encoding a model antibody were gene optimised and evaluated in stable cell lines. Optimisation of the model antibody resulted in increased mean antibody concentrations in stable cell lines. Antibody Concentration (mg/l) nonoptimised * Mean [Ab] (mg/l) optimised *Significant increase in mean [Ab] p<0.05 slide 31

32 Comparison of Vendor Genes optimised by different vendors can result in different levels of protein expression. 300 * ** 250 Antibody concentration (mg/l) Mean [Ab] (mg/l) Nonoptimised Vendor A Vendor B *Significant increase in mean [Ab] **Significant decrease in mean [Ab] slide 32

33 Conclusions Gene optimisation can be applied to increase levels of protein expression. A solution to problems related to gene sequence. Unlikely to solve issues where the protein sequence is not optimal. For manufacture of a recombinant protein, gene sequence is an important consideration when looking to generate commercially viable cell lines. slide 33

34 Enhancing Secretion

35 The Mammalian SRP cycle Nagai et al. (2003) EMBO J., 22: (A) SRP binds through the M domain of SRP54 to the signal sequence of membrane and secretory proteins emerging from the exit site of the large ribosomal subunit. (B) The Alu domain promotes transient arrest of the polypeptide chain elongation through an as yet unknown mechanism. The affinity of SRP54 for GTP increases upon docking of SRP with the ribosome. (C) The RNC-SRP complex diffuses to the ER membrane and docks with the SR mainly through the interaction between SRP54 and SRα in the GTP-bound form. SRβ in the GTP-bound form interacts with the RNC complex and induces the transfer of the signal peptide to the translocon. (D) SRP54 and SRα mutually activate their GTPases, and SRP dissociates from the SR upon hydrolysis of GTP, allowing the elongation of the polypeptide to resume. slide 35

36 Signal Sequences Antibody-derived signal sequences are often used for the production of recombinant antibodies. The amino acid composition of signal sequences can vary, but all have similar properties (e.g. hydrophobic core). It is not known how the features of a signal sequence influence the rate protein secretion. In order to evaluate if the choice of signal sequence contributes to increased antibody concentrations, nineteen alternative sequences were evaluated. Transient expression system. Stable cell lines. slide 36

37 Signal Sequences Use of vectors employing alternative sequences V1 to V19 in a transient expression system resulted in mean antibody concentrations in the range of 1.1 to 4.9 µg/ml. Five candidate sequences were selected for evaluation in stable cell lines. 6 Antibody concentration (µg/ml) WT WT opt V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11 V12 V13 V14 V15 V16 V17 V18 V19 slide 37

38 Evaluation of signal sequences in stable cell lines Evaluation of the chosen sequences in stable cell lines identified that two resulted in increased mean antibody concentrations. Antibody concentration (mg/l) * * WT WT opt V11 V13 V15 V16 V18 * *Significant increase in mean [Ab] p<0.05 vs WT opt Mean [Ab] (mg/l) slide 38

39 Evaluation of Product Quality Are the signal sequences cleaved during the process of secretion and not present in the final product? Inappropriate processing can negatively impact on product quality. The antibody-derived signal sequences have been previously shown to be appropriately processed in multiple products. There have been reports that incorrect processing can lead to increased aggregation. To determine if the antibody product generated using the alternative signal sequences were appropriately cleaved purified antibody was evaluated. SDS-electrophoresis. ESI-MS. slide 39

40 Evaluation of Product Quality Antibody generated using the alternative signal sequences was equivalent to the material generated the antibody derived sequences (WT opt ). SDS-electrophoresis (reduced) ESI-MS WT opt Light chain Lower marker System peaks Heavy chain Upper marker Molecular Weight (deglycosylated) WT opt KDa V16 V18 V KDa V KDa slide 40

41 Conclusions Choice of signal sequence can affect the level of antibody production. Equivalent product quality was observed indicating that there is no deleterious effect on the product when using the alternative sequences. The increase observed was in addition to that seen with gene optimisation. Modulating different aspects of the protein production pathway can result in additive benefits in recombinant protein production. slide 41

42 Comparison of Antibody Isotypes

43 Antibody Isotype and Expression Antibodies of the same isotype can have dramatically different expression levels. The sequence of the variable region is usually selected by the target antigen. The choice of constant region is most likely to be determined by the desired effector function for the molecule. It is possible that antibody isotype should be a consideration when attempting to maximise product yield. slide 43

44 Comparison of Antibody Isotype To evaluate this, GS vectors encoding a model antibody were generated where the heavy chain was either an IgG 1 or IgG 4 constant region. Stable CHO cell lines were generated using CHOK1SV host cells. 60 cell lines were selected using Lonza s cell line selection strategy and evaluated in suspension shake-flask culture in a fed-batch process designed to mimic laboratory scale bioreactors. slide 44

45 Comparison of Antibody Isotype Mean antibody concentrations were equivalent when the model antibody was expressed as either an IgG 1 or IgG 4 (p=0.178). Productivity of the lead cell line for both isotypes was the identical (2.4 g/l) Antibody concentration (mg/l) Mean [Ab] (mg/l) IgG 1 IgG 4 slide 45

46 Conclusion Use of an IgG 1 or IgG 4 isotype results in equivalent antibody productivities. Choice should be defined by the desired effector function for a given product. Variable region sequences may be the cause for the variation in antibody productivities for antibodies of identical isotype. slide 46

47 Summary All aspects of the protein production pathway are important for maximising recombinant protein expression. Expression platform. Gene sequence. Protein sequence. Choice of gene and protein sequence, along with use of leading expression technologies and process development are crucial in the design of commercially viable manufacturing processes. slide 47

48 Acknowledgments Lonza Biologics plc Cell Culture Process Development Tabitha Bullock, Rhys Davies, Salma Debar, Stephan Kalwy, Hilary Metcalfe, Alison Porter, Helen Rogers, Andy Racher, Rachel Sharpe, James Rance. Analytical Development Richard Aldcroft, Lisa Newey. slide 48

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