Case 1:18-cv-00924-CFC Document 376-2 Filed 09/27/19 Page 1 of 96 PageID #: 28774
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`C.A. No. 17-1407-CFC
`(CONSOLIDATED)
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`IN THE UNITED STATES DISTRICT COURT
`FOR THE DISTRICT OF DELAWARE
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`GENENTECH, INC. and CITY OF HOPE, )
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`Plaintiffs,
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`AMGEN INC.,
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`Defendant.
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`____________________________________)
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`GENENTECH, INC.,
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`Plaintiff and
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`Counterclaim Defendant,
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`AMGEN INC.,
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`Defendant and
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`Counterclaim Plaintiff.
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`____________________________________)
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`C.A. No. 18-924-CFC
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`APPENDIX TO GENENTECH’S LETTER-BRIEF CONCERNING
`CONSTRUCTION OF “FOLLOWING FERMENTATION” AND
`SUPPORTING DECLARATION OF DR. HANSJÖRG HAUSER
`
`
`
`

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`Case 1:18-cv-00924-CFC Document 376-2 Filed 09/27/19 Page 2 of 96 PageID #: 28775
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`U.S. Patent No. 8,574,869
`Bruce Alberts et al., MOLECULAR BIOLOGY OF
`THE CELL, Chapter 3 (4th Ed. 2002) (“Molecular Biology of
`the Cell”)
`Mullan et al., Disulphide bond reduction of a
`therapeutic monoclonal antibody during cell culture
`manufacturing operations, BMC Proc., 22(5) Suppl
`8:P110 (2011) (“Mullan 2011”)
`Trexler-Schmidt et al., Identification and Prevention of
`Antibody Disulfide Bond Reduction During Cell Culture
`Manufacturing, Biotechnol Bioeng., 106(3):452-61 (2010)
`(“Trexler-Schmidt 2010”)
`Kao et al., Mechanism of Antibody Reduction in Cell
`Culture Production Processes, Biotechnol Bioeng.,
`107(4):622-32 (2010) (“Kao 2010”)
`Mun et al., Air Sparging for Prevention of Antibody
`Disulfide Bond Reduction in Harvested CHO Cell
`Culture Fluid, Biotechnol Bioeng., 112(4):734-42
`(2015) (“Mun 2015”)
`Hutterer et al., Monoclonal Antibody Disulfide
`Reduction During Manufacturing, MAbs., 5(4):608-13
`(2013) (“Hutterer 2013”)
`Chung et al., Effects of Antibody Disulfide Bond
`Reduction on Purification Process Performance and
`Final Drug Substance Stability, Biotechnol Bioeng.,
`114(6):1264-1274 (2017) (“Chung 2017”)
`Fahrner et al., Industrial Purification of Pharmaceutical
`Antibodies: Development, Operation, and Validation of
`Chromatography Processes, Biotechnology and Genetic
`Engineering Reviews, 18:1, 301-327 (2001) (“Fahrner 2001”)
`Birch and Racher, Antibody production, Advanced Drug
`Delivery Reviews 58(5-6):671-85 (2006)
`Webster’s Dictionary, “Fermentation”
`FDA Biotechnology Inspection Guide (excerpts)
`Persson et al., Mammalian Cell Fermentation, Production of
`Biologicals from Animal Cells in Culture (1991) (“Persson
`1991”)
`
`Appx1
`Appx96
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`Appx127
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`Case 1:18-cv-00924-CFC Document 376-2 Filed 09/27/19 Page 3 of 96 PageID #: 28776
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`Bödeker et al., Production of recombinant factor VIII from
`perfusion cultures: I. Large-scale fermentation, Animal
`Cell Technology (1994) (“Bödeker 1994”)
`Ozturk et al., Real-time Monitoring of Protein Secretion in
`Mammalian Cell Fermentation: Measurement of Monoclonal
`Antibodies Using a Computer-Controlled HPLC System
`(BioCad/RPM), Biotechnol Bioeng., 48:201-206 (1995)
`(“Ozturk 1995”)
`Kemp G., O’Neil P., Large-Scale Production of Therapeutic
`Antibodies: Considerations for Optimizing Product Capture
`and Purification, in: Subramanian G. (eds) Antibodies (2004)
`(“Kemp 2004”)
`Dwivedi, Validation of Cell Culture-Based Processes and
`Qualification of Associated Equipment and Facility, in:
`Ozturk, Cell Culture Technology for Pharmaceutical and Cell-
`Based Therapies (“Dwivedi 2006”)
`US 2007/0141687 (Porro)
`Kaufmann et al., Influence of low temperature on productivity,
`proteome and protein phosphorylation of CHO cells,
`Biotechnol Bioeng., 63(5): 573-582 (1999)
`Matijasevic et al., Hypothermia causes a reversible, p53-
`mediated cell cyle arrest in cultured fibroblasts, Oncol Res.,
`10(11-12): 605-610 (1998)
`Roobol et al., ATR (ataxia telangiectasia mutated- and Rad3-
`related kinase) is activated by mild hypothermia in
`mammalian cells and subsequently activates p53, Biochem J.,
`435(2):499-508 (2011)
`Hunt et al., Low-Temperature Pausing of Cultivated
`Mammalian Cells, Biotechnol Bioeng., 89(2):157-63, (2004)
`Yoon et al., Effect of Low Culture Temperature on Specific
`Productivity and Transcription Level of Anti-4-1BB Antibody
`in Recombinant Chinese Hamster Ovary Cells, Biotechnol
`Prog., 19(4):1383-6 (2003)
`Roobol et al., Biochemical insights into the mechanisms
`central to the response of mammalian cells to cold stress and
`subsequent rewarming, FEBS J., 276(1):286-302 (2009)
`
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`Case 1:18-cv-00924-CFC Document 376-2 Filed 09/27/19 Page 4 of 96 PageID #: 28777
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`Supplement to Roobol et al., Biochemical insights into the
`mechanisms central to the response of mammalian cells to
`cold stress and subsequent rewarming, FEBS J., 276(1):286-
`302 (2009)
`McGraw-Hill Dictionary of Scientific and Technical Terms
`(6th ed.), “Fermentation”
`Deposition of Jeffrey John Chalmers (excerpts)
`Amgen 2011 Annual Report and Financial Summary
`(excerpts)
`Deposition of Stuart Watt (excerpts)
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`Appx402
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`Case 1:18-cv-00924-CFC Document 376-2 Filed 09/27/19 Page 5 of 96 PageID #: 28778
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`502
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`A. Roobol and others
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`is consistent with the response to hypothermia being due to
`stabilization of p53 consequent to its phosphorylation at SerH
`and this increased level of 1353 then inducing p21 expression
`
`The ATR protein kinase regulates phosphorylation of p53 at Ser15
`upon exposure to mild hypothermia
`
`Having established that p53 is phosphorylated at Ser15 in response
`to mild hypothermia, we set out
`to establish the kinase(s)
`responsible for this phosphorylation. Phosphorylation at SerH of
`p53 can be mediated by several protein kinases, including ATM,
`ATR, DNA—PK and the stress response signalling pathway protein
`kinase p38MAPK (p38 mitogen—activatcd protein kinase) [26]. To
`determine whether any of these was effecting p53 phosphorylation
`during mild hypothermia, we used a combination of general and
`specific protein kinase inhibitors and siRNA knockdown. Initially,
`we used caffeine, a well known, although not very specific,
`inhibitor of the PIKK family of protein kinases [27]. In the
`concentration range usually employed (low millimolar) it inhibits
`both ATM and ATR, but DNA—PK is relatively resistant. However,
`another PIKK family member, mTOR (mammalian target of
`rapamycin), a protein kinase that positively regulates protein
`synthesis in response to nutrient availability and growth factor
`signalling, is also inhibited by low millimolar concentrations of
`caffeine [27]. This must be taken into account when assessing
`the effect of caffeine on hypothermia—induced p21 expression.
`In the short term, 2.5 leI caffeine inhibited phosphorylation
`of p53 at Ser15 when cells were transferred to 32"C, but had
`little effect when cells were transferred to 27”C (Figure 2A).
`During longer—term exposure to caffeine, phosphorylation of p53
`at Ser15 was less sustained than in the absence of caffeine and
`p21 expression was reduced, under both hypothermic conditions
`investigated (32 and 27 0C) (Figure 2B). When compared with
`the inhibition of general protein synthesis by caffeine (due to
`mTOR inhibition), the inhibition by caffeine of p21 expression
`was greater (Figure 2D), consistent with either ATM or ATR
`being involved in hypothermia—induced expression of p21. More
`specific inhibition of DNA—PK with NU7441 [28] had no effect
`on either hypothermia—induced phosphorylation of p53 at Ser15
`or induction of p21 (Figures 3A and 3B). Thus, of the potential
`PIKK kinases that could phosphorylate p53 at Ser” upon mild
`hypothermia, these results suggested that either ATM or ATR is
`responsible.
`a widely used,
`The
`fungal metabolite wortmannin is
`3—kinases,
`and
`irreversible,
`inhibitor of phosphoinositide
`treatment of cells with micromolar concentrations of
`this
`
`compound causes inhibition of ATM, DNA—PK and mTOR [29].
`However, ATR is relatively resistant to wortmannin, and cells
`require exposure to concentrations in excess of 100 MM before
`ATR is inhibited [29]. In agreement with the results from the
`caffeine studies, which suggested that ATR might phosphorylate
`p53 at Serls, 20 MM wortmannin had no effect on hypothermia—
`associated phosphorylation of p53 at Ser15 and marginally
`inhibited p21 induction (Figure 2C). However, in contrast with
`inhibition by caffeine, inhibition of general protein synthesis by
`wortmannin was not significantly different from inhibition of
`hypothermia—induced p21 expression by wortmannin (Figure 211').
`We then used a specific inhibitor of ATM, KU0055933 [28], and
`this inhibited neither hypothermia—associated phosphorylation of
`p53 at Ser” nor induction of p2l (Figures 3A and 3B). Therefore,
`using specific inhibitors to DNA—PK and ATM, we were able to
`demonstrate that neither is the primary kinase involved in the
`hypotherniiaiindueed p537p2l pathway.
`
`© The Authors Journal compilation © 2011 Biochemical Society
`
`Although these inhibitor data are consistent with a signalling
`pathway in which ATR is a key kinase in the hypothermia—induced
`p53—p21 pathway, they are not specific ATR inhibitors, therefore,
`to test this hypothesis further, siRNA knockdown of ATR mRNA
`was employed. This approach has been shown to effectively
`reduce ATR protein levels by approx. 70 % 24 h after transfection
`[30,31] and therefore, although this does not obliterate protein
`levels, a knockdown would be expected to result in decreased
`Sens—phosphorylated p53 in response to mild hypothermia if
`this kinase is responsible. Two commercial validated siRNAs to
`human Al‘R were tested for their ability to knock down CHO—Kl
`ATR mRNA due to the lack of availability of such reagents for
`CH0 ATR. As expected, both siRNAs efficiently decreased HeLa
`cell ATR mRNA over a 48 h period by between 67 and 77%
`(Figure 4A). When tested in CHO—K1 cells, exposure to one of
`these siRNAs for 48 h decreased CHO ATR mRNA by 77787 %.
`However, knockdown by the second siRNA was less effective
`and more variable in CHO cells (Figure 4A). Knockdown of ATR
`mRNA was maintained at 72 h and,
`to a lesser degree, at 96 h
`post—transfection (Figure 4A). We confirmed that knockdown of
`ATR mRNA resulted in a knockdown in ATR protein levels in
`both HeLa and CIIO cells by Western blotting, which showed
`that ATR protein levels were reduced by 55—85 % after a 48 h
`exposure to ATR siRNA (Figure 4B). Following transfection
`with these siRNAs, cells were maintained at 370C for 48h
`before transfer to either 32°C or 27°C for a further 10 h. The
`
`decreases in ATR mRNA and protein observed after a 48h
`exposure to ATR siRNA were clearly mirrored by the decrease in
`the extent of phosphorylation at Ser15 of p53 under these mildly
`hypothermic conditions (Figure 4C). Inhibition of cold—induced
`phosphorylation of p53 at Ser” was still evident at 72 h, but not
`at 96 h, post—transfection (Figure 4D), but at this last time point
`the hypothermia—associated phosphorylation of p53 was already
`in decline (Figures 1B and 4D). These results are consistent
`with the inhibitor data indicating that hypothermia induces p53
`phosphorylation and p21 activation through the ATR7p537p21
`signalling pathway. Furthermore, the relative longevity (several
`days) of p53 phosphorylation at Ser15 during hypothermia is also
`consistent with this phosphorylation being regulated by ATR [3 2].
`we note that although knockdown of ATR protein was clearly
`achieved, ATR protein was still present and some phosphorylated
`p53 was also present in the knockdown experiments (Figures 4C
`and 4D). We were unable to ascertain from these results whether
`the phosphorylated p53 present upon cold shock was due to the
`residual ATR protein present or as a result of an additional
`pathway not
`investigated in the present study. Despite this,
`when cells were shifted to 27°C following knockdown of ATR
`for 48 h at 37”C by siRNA, those wells in which knockdown
`had been undertaken initially showed an increased in cell
`numbers 1 and 2days after being placed at 27”C above that
`observed in the mock knockdown (see Supplementary Figure
`81 at http://wwaiochemJ.org/bj/435/bj4350499add.htm). This
`further suggests that p53 activation through ATR is involved in
`the inhibition of cell proliferation upon cold shock at 27 DC. This
`effect was lost after 2days at 270C, probably because, at this
`stage. the knockdown cells at a higher cell concentration are
`beginning to experience nutrient and growth stresses that lead
`to a decrease in cell number as seen in Supplementary Figure S l.
`
`Involvement of the p38MAPK stress kinase signalling pathway in cell
`cycle arrest during mild hypothermia
`
`Although our results show that ATR is involved in the regulation
`of p53 Ser15 phosphorylation upon mild hypothermia and rule out
`
`CONFIDENTIAL
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`GNE-HER_002943009
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`Appx363
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`

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`Case 1:18-cv-00924-CFC Document 376-2 Filed 09/27/19 Page 6 of 96 PageID #: 28779
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`ATR and p53 activation during the response to mild hypothermia
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`Figure 2 Caffeine inhibits both phosphorylation of p53 at Ser‘5 and p21 induction associated with mild hypothermia, butwortmannin does not
`
`(A) Immunoblot detection of p53 phosphorylated at ’Serl5 and total p53 protein in lysates of CHO—K1 cells. with orwithout pre—treatment with 2.5 mM catteine for 30 min at 3.700 immediately prior
`to exposure to the indicated temperatures tor the indicated number of hours. (B) lmmunoblot detection 0t p53 phosphorylated at Ser“, total p53 protein, p21 and fl-actin in lysates 0t CHO-K1 cells,
`with or without pretreatment with 2.5 mM catteine tor 30 min at 37 0 0 immediately prior to exposure to the indicated temperatures for the indicated number of days. (C) Immunoblot detection of p53
`phosphorylated at Ser‘5 p2t and {I—actin in lysates of CHO-K1 cells exposed to 20 MM wortmannin for 30 min at 37% prior to incubation for the indicated times at 27 DC or 32 OC. The response
`0t total p53 protein levels tor these time points at 27 C C and 32 0C are shown in (B). (D) Quantification ot the inhibition by 2.5 mlvl caffeine of general protein synthesis and 0t hypothermia—induced
`p21 expression. (E) Quantitication ot the inhibition by 20 MM wortmannin of general protein synthesis and ot hypothermiaeinduced p21 expression.
`
`ATM and DNA—PK, this phosphorylation could also be effected by
`the stress response protein kinase p3 8WK (Hogl in yeast), either
`directly [33] or through its phosphorylation at Ser33 and Ser46 of
`p53 that, in turn, enhances phosphorylation at Ser" [34]. In yeast,
`this protein kinase is activated by osmotic stress or exposure to
`cold [35], whereas in mammalian cells, it has also been shown
`to be activated by osmotic stress [36]. p38MAPK is also activated
`by hypoxia and it has been reported that mildly hypothermic
`mammalian cells are hypoxic [37]. Furthermore, ATR can also
`phosphorylate. and thereby activate, p38MAPK [38]. It was therefore
`considered important to determine Whether the p381“APK protein
`
`kinase was involved, either independently, or through activation
`by ATR, in the p53—p21 pathway induced by mild hypothermia.
`SP203580 is an inhibitor
`frequently used for assessing
`involvement of p38MAPK in signalling pathways [39]. Although this
`inhibitor can also inhibit casein kinase 1 [5], this kinase will not
`phosphorylate p53 at Ser15 [40], therefore this inhibitor allowed
`us to investigate potential p38MAPK involvement in hypothermia—
`induced phosphorylation of p53 Serls. Treatment of CHO—K1
`cells with 10 MM SP203580 for 30 min prior to transfer to
`27°C or 32°C reduced both phosphorylation at Ser15 of p53
`and expression of p21 at these temperatures (Figure 5A). Since
`
`© The Authors Journal compilation © 2011 Biochemical Society
`
`CONFIDENTIAL
`
`GNE-HER_002943010
`
`Appx364
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`

`

`Case 1:18-cv-00924-CFC Document 376-2 Filed 09/27/19 Page 7 of 96 PageID #: 28780
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`504
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`A. Roobol and others
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`no inhibitor
`
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`
`Specific inhibitors of DNA-PK and ATM do not abrogate the cold-
`Figure 3
`induced phosphorylation of p53 and induction of p21
`
`CHO—K1 cells were grown at 37°C for 24 h, then 1 (.LM DNA—PK inhibitor NU7441 or 10 MM
`ATM inhibitor KU0055933 was added as indicated. Aiter a further 30 min of incubation at 37 ° C,
`cells were either maintained at 37°C or transferred to 32°C or 27°C for the indicated number
`01 hours. immunoblots 01 cell lysates were probed for Ser‘S-phosphorylated p53 (A) p21 (B)
`and fi—actin (C). Total p53 protein levels at these temperatures (4, 48 and 96 h) were established
`previousiyand reported in Figures 1 and 2.
`
`SP20358O had no effect on general protein synthesis (Figure 5B),
`its inhibition of p21 expression suggested involvement of p38MAPK
`in the hypothermia—induced p53—p21 pathway.
`To determine whether this p38 mechanism was a second
`pathway leading to phosphorylation at Ser15 of p53 independently
`of the ATR route, treatment with SP203580 was combined with
`siRNA knockdown of ATR. The resulting effects of combined
`ATR knockdown and SP203580 treatment on hypothermia
`induced phosphorylation at Ser15 of p53 and the p53 isoform
`pattern (Figure 6) mirrored those effects observed for ATR
`knockdown alone (Figure 4). This suggests that the involvement
`of p38MAPK in hypothermia—induced cell cycle arrest lies within,
`rather than acts independently of, the ATR pathway; otherwise,
`the effects of ATR knockdown and SPZO'SSSO treatment should
`
`have been additive. Therefore we suggest that the p38MAPK protein
`kinase is involved in phosphorylation of p53 at Ser15 upon mild
`hypothermia through activation by ATR.
`
`How is ATR activated upon exposure of CHD-K1 cells to mild
`hypothermia?
`
`Our results confirmed that ATR is activated upon CHO—K1 cells
`being exposed to mild hypothermia, which in turn phosphorylates
`Ser15 of p53 and p21 induction. However, how might ATR itself be
`activated upon mild hypothermia? We used immunofluorescence
`to determine whether there was any change in the localization
`of ATR following cold shock (see Supplementary Figure S2 at
`http:”wwaiochemJ.org/bj/435/bj4350499addhtm). Using this
`approach, it was found that at 2—48 h after cold shock at 27 OC,
`ATR appeared
`to
`be
`concentrated into
`the
`nucleolus
`(Supplementary Figure S2). We also noted an overall increase
`
`© The Authors Journal compilation © 2011 Biochemical Society
`
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`siRNA knockdown of ATR mRNA inhibits hypothermia—induced
`Figure 4
`phosphorylation of p53 a13er‘5
`
`(A) HeLa and CHO—K1 cells were transtected with SnM siRNAs against human ATR
`mRNA and then maintained at 37°C for 48—96h beiore undertaking qRT—PCR analysis
`01 ATR mRNA levels
`in
`total RNA.
`(B)
`lmmunoblot detection of ATP in HeLa and
`CHO-K1 cell
`iysates prepared atter 48—96h of exposure to ATR stRNAs at 37°C (m,
`mock transiected; 1, ATR siRNA 1; 2, ATR siRNA 2).
`(C)
`lrnmunoblot detection 01 total
`p53 protein and p53 phosphorylated at Seri5 in cell
`lysates of CHOAK1 cells 48h after
`siRNA knockdown 01 ATE mRNA at 37°C lollowed by 10h at 27°C or 32°C (ut,
`untreated;
`in. mock transfected; 1, ATR siRNA 1; 2, ATR siRNA 2).
`(D)
`inhibition of phos-
`phorylation of p53 at Seri5 is maintained over longer periods of siRNA knockdown (kd) of ATR
`than 48 h. CHO-K1 cells were transtected with siRNA 1 or mock transtected (m), incubated at
`37°C ior 72 h or 96 h and then maintained at 37°C or transierred to 27°C for a further 10 h
`prior to extraction for immunoblot detection of the indicated proteins.
`
`in ATR—associated fluorescence throughout the cell, particularly
`between 6 and 24 h of exposure to 27 0C.
`In addition to localization studies, we investigated changes to
`the lipid content of cold—shocked cells, When prokaryotic and
`lower eukaryotic cells are exposed to hypothermic conditions,
`the unsaturated fatty acyl content of cell membrane lipids has
`been reported to increase [14]. In mammalian cells, exposure
`to the Ca2 rindependent phospholipase A2 inhibitor BEL at
`37°C also increases the unsaturated fatty acyl content of
`phosphatidylcholines and activates ATR [17]. We therefore
`compared the effect of BEL treatment with that of hypothermia
`011 cellular lipid composition to determine whether a similar
`effect could be observed that might offer an explanation of ATR
`activation upon mild hypothermia. To achieve this, MS analysis of
`total lipids extracted from cells maintained at normal temperature
`(37°C), after treatment with BEL, and at mildly hypothermic
`temperatures was performed. Multivariate analysis (PCADFA) was
`applied to the resulting data with crossevalidation as described
`
`CONFIDENTIAL
`
`GNE-HER_002943011
`
`Appx365
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`

`

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`+ 10 mM SP20358O
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`ATR and p53 activation during the response to mild hypothermia
`
`505
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`p21 expression
`
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`inhibitor SP20358I]
`p38”““’"
`The
`Figure 5
`associated phosphorylation of p53 at Ser‘5 and p21 induction
`
`(A) CHO-K1 cells were exposed to 10 MM SP2Ci358Ci for 30 min at 37 C C and then transferred to
`27 0C or 32°C for the indicated times (1724 h). lmmunoblot detection of p53 phosphorylated
`at Ser‘S; total p53 protein and p21 with fi—actin as an indicator of protein loading is shown.
`(B) Quantification of
`the effects of 10 MM SP203580 on general protein synthesis and
`hypothermiaeinduced p2i expression
`
`ep53
`
`p53
`
`
`
`
`
`
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`_
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`utm 12utm1 2 utm12
`37°C
`32‘3C
`27°C
`
`Inhibition of hypothermia-induced phosphorylation of p53 at Ser15
`Figure 6
`by ATR knockdown is not increased by additional inhibition of p38"’“lPK
`
`At 48 h aftersiPNA-mediated ATR mRNA knockdown; CHO—K1 cells were additionally exposed to
`SP203580 for 30 min at 37 0C then transferred to 27 0C or 32 0C for a furllier 10 li. lmmunoblot
`detection of total p53 protein; p53 phosphorylated at Serl5 and fieactin in lysates of CHOeKl
`cells treated in this way is shown. ut; untreated; m; mock transfected; 1; ATP! siRNA 1; 2; ATR
`isNA 2 as in Figure 4.
`
`Discriminant function l
`
`Exposure of CHO-K1 cells to mild hypothermia is associated with
`Figure 7
`changes in the cellular lipid profile
`
`PCeDFA of all samples. Upper panel: PCeDFi plotted against PCeDF2. Lower panel: PCeDFl
`plotted against PC-DFB. The first ten PCs were used by the DFA algorithm and this accounted
`for 99.8% of the total explained variance. The multivariate model was constructed using three of
`six samples in each class (no asterisk) and crossfiralidated by projection of the remaining three
`samples (shown with an asterisk). The level of agreement of the samples projected with those
`used to construct the model highlight that the model is validated. Class 1; control maintained at
`37 0 C for 6 h with no treatment; Class 2; control maintained at 37 C C for 6 hwlth BEL treatment;
`Class 3; maintained at 2750 for 6 h; Class 4; maintained at 32“C for 6 h; Class 5; recovery
`at 37 DC or 2 h after a temperature of 27 DC for 6 h; Class 6; recovery at 37% for 2 if after a
`temperatLre of 32 ”C for 6 h.
`
`
`
`in the Experimental section and shown in Figure 7. The results
`show hat BEL—treated and 37°C control cells were different
`from all cells cultured at reduced temperatures, and the chemical
`treatment and control cells dominated the separation of the second
`canonical variate (Figure 7, upper panel). When PC—DFl was
`plottec against PC—DF3 (Figure 7, lower panel), each class was
`biolog'cally distinct from other classes, highlighting the fact that
`the detectable lipid profile of each of the six classes was different
`and perturbations (chemical or temperature—based) resulted in
`distinct phenotypic changes.
`Furt 1er1nore, univariate analyses using Kruskal—Wallis analysis
`of variance to define the lipids that were statistically significantly
`changing
`(see Supplementary Table
`81
`at
`http://www.
`Biochem].0rg/bj/435/bj4350499add.htm)
`revealed
`that
`the
`positive control
`(treatment with BEL)
`showed a different
`relative change to the control
`in the PC—DFA model when
`compared with the temperaturertreated cells. Ten lipids were
`statistically different (P < 0.05) and all showed an increase in
`
`CONFIDENTIAL
`
`GNE-HER_002943012
`
`@i The Authors Journal compilation © 2011 Biochemical Society
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`

`Case 1:18-cv-00924-CFC Document 376-2 Filed 09/27/19 Page 9 of 96 PageID #: 28782
`
`506
`
`A. Roobol and others
`
`their relative concentration in the BEL—treated cells compared
`with the control. Cells treated at 27 UC and 32 UC (mild
`hypothermia)
`for 6 h showed a similar
`trajectory from the
`control samples, with the 270C samples showing a greater
`biological difference in multivariate space compared with the
`samples perturbed at 32 ° C. However, more statistically significant
`changes were observed between control and 32°C samples in
`the univariate analysis (37 compared with four changes for
`32°C and 27°C respectively). All of the changes showed an
`increase in concentration of a range of lipids, predominantly
`phospholipids (diacylglyceroserines, diacylglyceroinositols and
`diacylglycerophosphocholines).
`In most cases, although not
`exclusively, an increase in the unsaturated double—bond content
`was present in the lipids of increased abundance This highlights
`a definitive increase in the production of a specific class of lipids
`in response to temperatureibased perturbations. The increase in
`temperature after hypothermia perturbation (recovery) provides
`a change in the lipid profile from that at reduced temperature,
`but this lipid profile is distinct from all other samples. This
`shows that an increase in temperature changes the lipid profile,
`but not
`to a normal profile at 2h after the return to 37UC.
`Decreases in the relative concentration of lipids were observed
`in the change from lower to higher temperature, of the same
`classes of lipids that were observed to increase as the temperature
`was decreased. This highlights the specific role of these lipids in
`the response to temperature perturbation and how their relative
`concentration is temperature dependent. Although many of the
`lipids were chemically identified, we were unable to show
`significant changes in the overall unsaturated fatty acyl content
`of cell membrane lipids. However, we have shown an increase in
`polyunsaturated lipids upon mild hypothermia consistent with a
`previous study showing that an increase in phosphatidytcholines
`containing polyunsaturated fatty acids
`activates ATR—p53
`signalling at 37°C [17].
`
`DISCUSSION
`
`Although we [12] and others [10,13] have documented that p53
`activation of p21 is a key mechanism by which mammalian cells
`initiate cell cycle arrest upon being subjected to mild hypothermic
`temperatures, the mechanism by which p53 is activated and the
`cellular mechanisms that allow the perception of cold and
`subsequent activation of p53 have remained undetermined. In
`the present study, we have shown that the exposure of CHO—
`K1 cells to mildly hypothermic conditions activates the ATR
`kinase that subsequently activates p53 by phosphorylation at Ser15
`and hence the ATR—p53—p21 signalling pathway. We note that
`although our experiments clearly show ATR regulation of p53
`phosphorylation upon cold shock, in our ATR knockdown and
`inhibitor experiments some ATR protein and phosphorylated p53
`still remained and we were unable to ascertain from these results
`
`whether the phosphorylated p53 present upon cold shock in these
`experiments was due to the residual ATR protein present or a
`result of an additional signalling pathway not investigated in the
`present work.
`We speculate that the primary stimulus for the activation of the
`ATR—p53—p2l signalling pathway upon mild hypothermia may
`be changes in membrane rigidity [14] as a direct result of changes
`in membrane lipid composition (horneoviscous adaptation). Our
`results show changes in the levels of polyunsaturated fatty acids
`upon cold shock that are known to influence the fluidity of cellular
`membranes, and, furthermore, that these changes correlated with
`the activation of ATR. As described above, a previous study has
`demonstrated that changes to cell membrane fluidity and increased
`polyunsaturation activates ATR and the authors of that study
`
`© The Authors Journal compilation © 2011 Biochemical Society
`
`suggest that this occurs as a result of ATR ‘sensing’ the change in
`the ratio of polyunsaturated to saturated hydrocarbons [17]. The
`question is how might this change in lipid composition activate
`ATR? Zhang et a1. [17] suggest that this is the result of changes
`in the fluidity and function of the nuclear envelope whereby the
`nuclearilocalized ATR senses these changes and is activated. We
`speculate further that this leads to an intranuclear relocalization of
`ATR upon activation (as shown in Supplementary Figure S2), p53
`activation and cell cycle arrest. Such intranuclear relocalization
`of ATR to nuclear foci has been documented in response to both
`hypoxia [41] and DNA damage [42]. The overall increase in ATR—
`associated fluorescence throughout the cell during early exposure
`to 27 OC without an increase in immunoblot detection of ATR also
`suggests that, additionally, there may be a conformational change
`in ATR upon exposure of the cell to cold that renders the protein
`more accessible to the antiiATR antibody used.
`CHOiKl p53 carries a single point mutation at codon 211 in
`exon 6 in the DNA—binding domain of the molecule, although this
`mutation is not within an evolutionarily conserved region [43].
`Furthermore, CHO—Kl p53 is rather more abundant and stable
`than wild—type p53. At 37 “C, its half—life is 5.2 h [12] compared
`with the more usual range of 20—60 min for p53 half—lives.
`Furthermore, CHO—Kl p53 is not stabilized further, and thereby
`increased in amount, by ionizing radiation,
`i.e. by the ATM
`signalling pathway alone [43]. Thus, even though CHO—Kl p53 is
`relatively abundant, it is not sufficient, under normal conditions,
`to activate transcription of p21. Even under mildly hypothermic
`conditions, when p21 transcription is activated. increases in CHO—
`K1 p53 total protein are very modest (Figures 1, 2, 4 and 6).
`What does change markedly in response to hypothermia is the
`phosphorylation status ofp53. For wild—type p53, phosphorylation
`at Ser15 enhances p53 transactivation of p21 transcription by
`increasing the binding of p53 to its transcriptional co—activator,
`p300/CBP [44]. Furthermore, although phosphorylation at Ser15
`of p53 is not itself sufficient to disrupt the interaction between
`p53 and Mdm2 (murine double minute 2) that targets p53 for
`degradation, phosphorylation at this site is a prerequisite for
`phosphorylation at Ser20 of p53. Ser20 phosphorylation inhibits
`the binding of p53 to Mdm2 [45]. The overall effect of
`phosphorylation of Ser“ of wild—type p53 is therefore 2—fold,
`i.e. enhanced stability and enhanced transcriptional activation
`ability. In the context of CIIO—Kl cells, this must mean that
`phosphorylation at Ser15 is sufficient to enhance the transcription
`factor activity of an already a