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Gene amplification for high level production in CHO dhfr- cells.
  Reduction of folate to tetrahydrofolate

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                                            Reduction of folate to tetrahydrofolate
DNA
RNA                                                            FH4
                    IMP
                                                                 serine
                                              PurBios                     DHFR
      Precursors
                                                                                 DHFR
                                               CH   FH4    SHM            FH2             Folate

                                                     glycine                        TMP            DNA
                                               MTDH                        TS

                                                          CH2=FH4
                                                                                 dUMP




                                 DHFR:              Dihydrofolate reductase
                                 SHM:               Serine hydroxymethyltransferase
                                 TS:                Thymidylate synthetase
                                 PurBios:           Purine nucleotide biosynthetic pathway
                                 MTDH:              Methylenetetrahydrofolate dehydrogenase
Biosynthesis of glycine

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                                            Biosynthesis of glycine
DNA
RNA                                                    FH4
                          IMP
                                                         serine
                                      PurBios                     DHFR
          Precursors
                                                                         DHFR
                                       CH   FH4    SHM            FH2             Folate

                                             glycine                        TMP            DNA
                                       MTDH                        TS

                                                  CH2=FH4
                                                                         dUMP




                                DHFR:       Dihydrofolate reductase
                                SHM:        Serine hydroxymethyltransferase
                                TS:         Thymidylate synthetase
                                PurBios:    Purine nucleotide biosynthetic pathway
                                MTDH:       Methylenetetrahydrofolate dehydrogenase
                                                                                 4

                           Biosynthesis of thymidylate
DNA
RNA                                    FH4
          IMP
                                         serine
                      PurBios                     DHFR
      Precursors
                                                         DHFR
                       CH    FH4    SHM           FH2             Folate

                             glycine                        TMP            DNA
                       MTDH                        TS

                                   CH2=FH4
                                                         dUMP




                DHFR:       Dihydrofolate reductase
                SHM:        Serine hydroxymethyltransferase
                TS:         Thymidylate synthetase
                PurBios:    Purine nucleotide biosynthetic pathway
                MTDH:       Methylenetetrahydrofolate dehydrogenase
                                                                                 5

                    Biosynthesis of purine nucleotides
DNA
RNA                                    FH4
          IMP
                                         serine
                      PurBios                     DHFR
      Precursors
                                                         DHFR
                       CH   FH4    SHM            FH2             Folate

                             glycine                        TMP            DNA
                       MTDH                        TS

                                  CH2=FH4
                                                         dUMP




                DHFR:       Dihydrofolate reductase
                SHM:        Serine hydroxymethyltransferase
                TS:         Thymidylate synthetase
                PurBios:    Purine nucleotide biosynthetic pathway
                MTDH:       Methylenetetrahydrofolate dehydrogenase
                                                                                6

DHFR-deficient cells require glycine, thymidine and a purine

DNA                hypoxanthine
RNA
          IMP
                                     FH4
                                       serine
                     PurBios                    DHFR
      Precursors
                                                        DHFR
                      CH   FH4    SHM           FH2              Folate

                           glycine                         TMP            DNA
                       MTDH                      TS

                                 CH2=FH4
                                                       dUMP
                                                                     TdR
                DHFR:      Dihydrofolate reductase
                SHM:       Serine hydroxymethyltransferase
                TS:        Thymidylate synthetase
                PurBios:   Purine nucleotide biosynthetic pathway
                MTDH:      Methylenetetrahydrofolate dehydrogenase
                                                 7



        Transfection strategies
1.   YFG linked to a dhfr minigene on a single
     plasmid
       A. ~Insures co-integration
       B. ~Insures co-amplification

2.   YFG and dhfr on separate plasmids
      A. Allows a high ratio of YFG to dhfr to
             start
                                                 8




                          dhfr
DHFR-
                                     YFG




         Select in
        purine-free
         medium




DHFR+
                  Increasing [MTX]


                                     DHFR+++++
                                     YFG +++++
                                                      9

   Co-amplification of genes on unlinked plasmids


              DHFR                   YFG



                            Transfection
                            Co-integration, usually

                   DHFR                  YFG




                            Step-wise
                            Low MTX --> High MTX
                            Co-amplification




Wigler et al., PNAS, 1980
                                                                                             10
Use a high ratio (e.g., 1000X) of YFG plasmid DNA to dhfr plasmid DNA

 DHFR         YFG         YFG          YFG          YFG         YFG      YFG        YFG



 YFG        YFG        YFG       YFG          YFG         YFG     YFG     YFG       YFG



                                             Transfect with 10 ug of YFG and 10 ng of dhfr
                                             Co-integration
                                             Multiple copies of YFG from the start



                                             Step-wise
                                             Low MTX --> High MTX
                                             Co-amplification




  (Wigler et al, Cell, 1979:X174 DNA)
                                                                    11

          Transfection with both genes in one vector
              and even in one transcription unit


                                 Y.F.G.   DHFR




                                 Y.F.G.   DHFR
Dicistronic mRNA




                                      Poor translation initiation
                                      Low DHFR
                                      Supersensitive to MTX
                                      Select initially in low MTX
                                      More room for amplification
 Kaufman, RJ, et al, NAR, 1993
                                                             12

Use a weak dhfr promoter to confer supersensitivity to MTX


                   DHFR                     YFG



                               Transfection,
                               Co-integration, usually

                        DHFR                    YFG



                               Stepwise
                               Very low MTX --> high MTX
                               Co-amplification
                               More room for amplification




 Wigler et al., PNAS, 1980
                                                                                              13
Use a high ratio (e.g., 1000X) of YFG plasmid DNA to dhfr plasmid DNA
and a poorly expressed dhfr minigene
 DHFR         YFG         YFG           YFG          YFG         YFG      YFG        YFG



 YFG        YFG        YFG        YFG          YFG         YFG     YFG     YFG       YFG



                                              Transfect with 10 ug of YFG and 10 ng of dhfr
                                              Co-integration
                                              Multiple copies of YFG from the start



                                              Stepwise
                                              Very low MTX --> high MTX
                                              Co-amplification




  (Wigler et al, Cell, 1979:X174 DNA)
                                                                                      14
                            Possible amplification protocol

Pool of transfectants
selected for growth
in purine-free medium   0            10         20            40       80
                                                                            nM MTX
                                                                            in -GHT
                                                                            medium


 Check                  10           20         40            80       160 nM MTX
 selected
 populations
 for Ig
 production



                        40           80         160           300     1000 nM MTX




                                                        Finally clone several
                                             etc.       for final stages
                      15




Papers:

Bredveldt
Waldemann
Herold

Kohler and Milstein
Littlefield
Wigler
Tanaka
Davies and Reff
Reff patent
                                                                     16

Position effect (Reff- IDEC)

Expression level is influenced by the position of integration
( in transgenic mice and transfected cells )

Euchromatin vs. heterochromatin, gradation, proximity of enhancers

Reff:
Screen for a high producer site among many transfectants.
Integrated gene is linked to 1/3 of a neo gene (3 exons),
and several selectable markers including dhfr (amplifiable) .
Use this transfectant as the host for YFG linked within (why?)
 the other 2/3 of the neo gene.
Overlapping neo sequences target homologous recombination.
Select for G418 resistance (reconstrcution of the neo gene)
Drug-resistant colonies carry YFG at the hot spot for production,
within an intron of the neo gene.

Homologous recombination frequency is low (10-7),
but you only need one good transfectant.
Amplify with MTX/dhfr.
                               17

Reff patent 2002 (IDEC)
Exploits position dependence
for high exprerssion level
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19
                         20




Low freq event < 10-6)
                                                            21




Ig promoters




               splicing




Mab genes are expressed from within the neo gene
Advantage? Tightly integrated, never lost in a neoR cell.
Disadvantage? Complex transcription, competition?
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  Sequential amplification (R. Kaufman)

  1) Dhfr + MTX

  2) Adenosine deaminase (ADA) + deoxycoformycin

  AAU medium :
  adenosine (requires ADA
  alanosine (inhibits purine synthesis)
  uridine (counteracts high adenosine toxicity –[obscure])
  +deoxycoformycin          (inhibits ADA – gradual increase of dose)


After a maximum is reached with MTX, bring in YFG again linked to ADA
                                                                   23

A different major system for high level Mab production

NS0 cells:
Mouse myeloma cells, high IgG producers  IgG- variants = NS0
No endogenous IgG, but cell is a natural IgG secretor.

Lack glutamine synthetase (GS):
glutamate + NH3 + ATP  glutamine + ADP + Pi

Vector = MAb genes driven by strong promoters (H-chain, L-chain)
+ GS cDNA gene (Bebbington).

Select on gln-free medium.

Inhibit GS with methionine sulfoximine (gln analog).

Select for GS overproducers.
(amplification of the GS cDNA gene and linked Mab genes)
Proprietary (Lonza Biologics).
                                                                 24
Do the math:


Reff: 55 pg/cell/day
Max cell density = 2 x 106/ml ?
So 2 x 109 cells/L
Therefore 55 x 10-12 g/cell/day x 2 x 109 cells/L =
110 x 10-3 g/L/day =
0.1 g/L/day

Lonza (contract manufacturer) claims best =
2.8 g/L yield
OK, if accumulate secreted protein for 28 days at 0.1 g/L/day.
(long, but not unreasonable)

30,000 L reactor:
30,000 L. X 2. 8 g/L. = 84 kg in 28 days,
x 12 = 1008 kg/year = 1,000,000 g/year

One MAb dose = 500 mg = 0.5 g
1,000,000/0.5 = 2 million doses per reactor per year.
6 doses per patient per year?
2,000,000/6 = 300,000 patients per year per reactor.
AT $10,000 per patient per year  $3B in sales
                25



Tanaka et al.
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Kohler and Milstein methods:
IEF
SRBC + complement colony assay
Allelic non-exclusion
Suggests using negative myelomas
High yield of antigen-reactive hybridomas (0.2, 3, 10%)
Generality: Other specialized cells? E.g., hepatoma-hepatocyte.
Neuroblastoma-neuron. Not much followed up.
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Wigler et al.:
Note uncloned total DNA used as a gene source (unusual)
Dhfr in A29 was altered as well as amplified (intrinsically somewhat MTX-resistant

				
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