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`1993/09/07
`
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`(51) -.INTL.CL. Cl2N-Ol5/12; CUQ-001/68; C071C-013/00; C07K-015/12·;_
`C07H-021/04; COlN-001/68
`
`(19) (CAJ APPLICATION FOR CANADIAN PATENT (12)
`
`(54) Microaomul Triglyceride Transfer Protein
`
`(72) Wetterau, John R., II -
`Sharp, Daru Y.
`-
`;
`Gregg, Richard E.
`-
`;
`
`(73) Same as inventor
`
`(30) (US) 847,503 1992/03/06
`
`(57) 32 Claime
`
`Hotica1
`
`Thl• •pplicatlon la •• tiled and may therefore contain an
`1ncocnplete apec:Ulcation.
`
`Canada -
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`CCA »'WttOwt•t PWDll·-. . .
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`Abstrut
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`MICROSOMAL TRIGLYCERIDE TRANSFER PROTEIN ·
`Nucleic acid sequences, partlaJlaffy ONA sequences.
`coding ror all or part of the high molecular weight subunit of
`mlcrosomaJ triglyceride transfer protein, expression vectors
`· containing the ONA sequences, host cells containing the ·
`expression vectors, and methods ulllzlng these materials. The
`Invention also concerns potypeptkle molecules comprising all or
`part of the high molecular weight subunit of microsomal
`trtgtyceride transfer protein, and methods for produdng these
`polypeptide molecules. The Invention eddltlonally concerns novel
`methods tor pr11venllng, ltablllzlng or causing regression ot
`atherosclerosis and therapeutic scents having such activity. The
`invention additionally concerns novel methods for lowertng seNm
`liquid levels and therapeUtlc agents having such activity.
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`MICROSOMAL TRIGLYCERIDE
`TRANSFER PROTEIN
`
`S
`
`Crg11-R1ftrtne• Ip Btl1t1d Appl!eat!pn
`This Is a continuation-in-part of U. S. patent application Ser.
`No. 847, 503, filed March 6, 1992.
`
`fleld pf tbt IDVJDtlgn
`This Invention relates to microsomal triglyceride transfer
`protein, genes for the protein, expression vedors comprising the
`genes, host cells comprislno the vedors, methods for producing
`the protein, methods for detecting Inhibitors of the protein, and
`methods of using the protein and/or Its Inhibitors.
`
`Background qt lbe lrwenllon
`The microsomal tritrlyceride transfer protEtln (MTP)
`catalyzes the transport of triglyceride (TG), cholesteryl ester (CE),
`and phosphatidylchollne (PC) between small unllamellar vesicles
`(SUV). Wenerau & Zltversmlt, Chem Phys l lpld5 38.. 205-22
`(1985). When transfer rates are ex?ressed as the percent of the
`donor lipid transferred per time, MTP expresses a distinct
`preferenee for neutral Hpld transport (TG and CE), relative to
`phospholipid transport. The protein from bovine liver has been
`Isolated and characterized. Wetterau & Zllversmlt, Chem Phys
`UJlll1i ail. 205-22 (1985). Polyacrylamlde gel eledrophoresls
`(PAGE) analysis of the purified protein suggests that the transfer
`protein Is a complex of two subunits of apparent molecular weights
`58,000 and 88,0oO, since a single band was pres."nt when
`purified MTP was electrophoresed under nondenaturing condition,
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`while two bands of apparent molecular weiohts 58,000 and
`&8,000 wore identified when electrophoresis was per1ormed In the
`prel"ence of Sodium dodecyl sulfate (SOS), These two
`polypej>tides are hereinafter referred to as 58 kOa and 88 kOa,
`respectiv~ly. or the 58 kDa and the 88 kOa component of MTP,
`respectively, or the low molecular weight subunit and the high
`molecular weight suounH of MTP, respectively.
`Characterization of the 58,000 molecular weight component
`of bovine MTP Indicates that It Is the .previously characterized
`1 O multlfunctlonal protein, protein disuffide lsomerase (POI).
`Wbt:erau al.Jd., J Biol Chem 265, 9800·7 (1990). The presence
`of PCI In the transter protein Is supported by evidence showino
`that (1) the amino terminal 25 amino adds ot the bovine 58,000
`kOa component of MTP Is Identical to that of bovine POI, and (2)
`disulfide· 1somerase activity was expressed by bovine MTP
`following the dissociation at the 51 kOa • 88 kOa protein complex.
`In addition, antibodies raised against bovine POI, a protein which
`by Itself has no TG transfer activity, were able to
`lmmunoprecipitate bovine TG transfer actlvl1)' tram. a solution
`containing purified bovine MTP.
`POI normally plays a role In the folding and assembly of
`newly synthesized disulfide bonded proteins within the lumen of
`the endoplasmic reticulum. Bulleld & Freedman, ~ .JJS, 649·
`51 (1988). It catalyze!'l the proper pairing ot cyste!ne residues Into
`disulfide bonds, thus catalyzing the proper folding of disulfide
`bonded proteins. In adrlitlon, POI has been reported to ~
`Identical to the beta subunit '>f human prolyl 4-hydroxylase. Koivu
`n.al.. J Bjol Chem 2§2, 6447·9 (1q&7) The role of POI in the
`bovine transfer protein Is not cl8ar. It does appear to be an
`essential component of the transfer protein as dissociation of POI
`from the 88 kOa component ot bovine MTP by either low
`concentrations of a denaturant (guanldlne HCI).. a chaotroplc ·
`agent (sodium perchlorate), or a nondenaturlng detergent (octyl
`glucoside) results In a loss of ~ansfer activity. Wetterau JIUJJ..
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`Blgchem!strv JQ. 9728·35 (1991 ). Isolated bovine POI has no
`npparent lipid transfer activity, suggesting that either !he 88 · kOa
`polypeptide Is the transfer protein or that It conlers lransler activity
`to the protein complex.
`The tissue and subcellular distribution or MTP activl!y In rats
`has been Investigated. Wetterau & Zilversmlt, Bjpchem Blopbys
`Adil 8Z5.. 610-7 (1986). Lipid transfer activity was found In liver
`and Intestine. Little or no transfer activity was found In plasma,
`brain, heart, or kidney. Within the liver, MTP was a soluble protein
`located within the lumen or the mlCRJsomal fraction.
`Approximately equal concentrations wera found In the smooth and
`rough mlcrosomes.
`Abatallpoprotelremla Is an autosomal recessive disease
`characterized by a virtual absence of plasma Hpoprotelns which
`contain apoDpoproteln B (apoB). Kane & Havel In The MetaOO!jc
`Basjs gf Inherited Q!sease. Sixth edition, 1139·64 (1989). Plasma
`TG levels may be as low as a few mg/dl, and they tall to rise after
`l'.it ln29stlon. Plasma cholesterol levels are often on~· 20-45
`mgldL These abnormalities are the rasuft or a genetic defect In
`the assembly andlor secretion of very low density llpoprotelns
`(VLDL) In the liver and chylomlcrons In the Intestine. The
`molecular basis for this defect has not been previously
`determined. In subjects examined, triglyceride, phospholipid, .and
`cholesterol synthesis appear normal. At autopsy, subjects are, tree
`of atherosclerosis. Schaefer .11.£, C!in Chem a!; 89-12 (1988).
`A link between the apoB gene and obetalipoproteinemia has been
`excluded In severai tamilies.-Talmud 11..BL. J cnn lnyest az ..
`1803-6 (1988) and Huang JILIL. Am J Hum Genet i6, 1141,8
`(1990).
`Subjects with abataRpoproteln;,,mla are afflicted with •
`numerous melacfles. Kane & Havel, J.W2.m. Subjects have tat
`malabsorptlon and.TG accumulation In their enterocytes and
`hepatocytes. Due to the absence of TG-rich plasma 6poproteins.
`there is a defect In the transport of fat-soluble vitamins such as
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`vitamin E. This resuhs In acanlhocytosls or erythrocytes,
`spinooerebellar a1axla wilh degeneration ol the lasclculus
`C\Jnealus and gracilis, peripheral neuropathy, degenerative
`pigmenlary retlnopathy, and oerold myopathy. Treatment of
`abetalipoproteinemic subjects Includes dietary restriction of tat
`intake and die'lary SUPJJl11mentation with vitamins A, E and K.
`To date, the physiological role of MTP has not been
`demonstrated. Jn dm. it catalyzes the transport of lipid molecules
`between phospholipid membranes. Presumably, it plays a similar
`role iO.l1m. and thus plays some role In lipid metabolism. The
`suboellular (lumen of the microsomal fraction) and tissue
`distribution (liver and ln!estlne) of MTP have led to speculation thal
`It plays a role In the assembly of plasma llpoprolelns, as these are
`the sites of plasma Hpaproteln assembly. Wetterau & Zllversmlt,
`15 Biocbem B!ophys AC1a m. 610-7 (1986). The ablllty of MTP to
`catalyze the transport of TG between membranes Is conslstenl
`with this hypothesis, and suggest~ that MTP may catalyze the
`transpol'I ot TG from Its site or synthesis In t'le endoplasmic
`reticulum (ER) membrane to r.asr.enl llpoproteln particles within
`the lumen of the ER.
`Ololsson and colleagues have studied llpoproteln assembly
`In HepG2 cells. BoSlrom .cJ.:al., J Big! Chem 263, 4434·42
`(1988). Their results suggest small precursor lipoprotelns become
`· larger with time. This would be consistent with the addition or
`transfer of llpld molecules lo nascenl lipoproteins as they are
`assembled. MTP may play a role In this process. In suppot1 or this
`hypothesis, Howell and Palade, J Cell Rjg! sz. 833-45 (1982),
`isolated nascent Opoprolelns from the hepatic Golgl traction or rat
`liver. Thera w:1s a spectrum of sizes of particles present with
`varying lipid and protein compositions. Pal'llcles of high density
`llpoproteln (HDL) density, yet containing apoB, were found.
`Higgins and Hutson, J t lpld Bas 25, 1295-1305 (1984), reported
`lipoproteins Isolated from Golgl wera consistently larger.than
`those from the endoplasmic retlculum, again su9ges1ing lhe
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`assembly of lipoproteins is a progressive event. However, there Is
`no direct evidence Jn the prior art demonstrating that MTP plays a
`role In llpld metabonsm or the assembly ol plasma llpoprotein.
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`Summ1rv pf the lnyentlo.n
`The present Invention concems an Isolated nucleic acid
`mole:ule comprising a nucleic acid sequence coding for all or part
`of the high molecular weight subunit of MTP andlor intron, S'. or 3'
`flanking regions thereof. Preferably, the nucleic acid molecule is a
`1 o ONA (deoxyr1bonuclelc acid) molecule, and the nucleic acid
`sequence Is a DNA sequence. Further preferred Is a nucleic acid
`havtng all or part of the nucleottc:le sequence as i.hown In SEQ. ID.
`NOS. 1. 2, 5, 7, 8, 1 together with 5, 2 togeth"r with 7, the first 108
`bases ol 2 together with 8, or the first 108 bases of 2 together with
`7and8.
`The present Invention also concerns a nucleic ac!d
`molecule having a sequence complementary :o the above
`sequences and/or lntron. 5', or 3' flanking regions thereof.
`The present invention further concerns expression vedono
`comprising a DNA sequence coding for au or part of the high
`molecular weight subunit of MTP;
`The present Invention additlonally concams prokaryotic or
`eukaryotlc host cells containing an expression vector that
`comprises a ONA sequence coding for all or part of the high
`25 molecular weight subunit of MTP.
`The present Invention additionally concerns polypeptlde
`molecules comprising all or part of the high molecular weight
`subunit of MTP. Preferably, the polypeptide ls the high molecular
`weight subunit of human MTP or the recombinanlly produced l:'ligh
`30 molecular weigh\ subunit or bovine MTP.
`The present Invention also concems methods for detecting
`nucleic acid sequences coding for all or part of the high molecular
`weight subunit of MTP or related nucleic acid sequences.
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`The present Invention ru11her concerns methods lor
`detecting an Inhibitor of MTP.
`The present Invention ru11her concerns a novel method for
`treatmont of atherosclerosis, or for lowering the level of serum
`lipids such as serum cholesterol, TG, PC, or CE In a mammalian
`species comprising administration of a therapeutically effective
`amount of an ,.gent that decreases the activity or amount of MTP.
`Such agents would also be useful for treatment of diseases
`associated or affected by serum lipld levels, such as pancreatitis,
`1 o hyperglycemia, obesity and the Uke.
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`Brief pescrlprlgn qt the Drawings
`Figure 1 shows bovine cDNA clones. The five bovine cONA
`Inserts are Illustrated. T.he continuous line at the top of the figure
`represonts the total cDNA sequence Isolated. Small, labeled bars
`above this Une map peptide and probe sequences. The open
`reading frame Is Indicated by the second One, followed by ••
`corresponding to 3' noncodlng sequences. Clone number and
`length are Indicated lo the left of each line repreHnting lhe
`corresponding region of the composite sequence. Clones 64 and
`76 were Isolated with probe 2A, clones 22 and 23 with probe 37 A
`and clone 2 with probe 19A. Eco RI Rnkers added ct Jring the
`cDNA library construction contribute the Eco RI restriction shes at
`the 5' and 3' ends of euch Insert. The Internal Eco RI site In ln~rts
`22 and 76 ls encoded by the cDNA sequence. The Nhe I
`restriction site was utiftzed in pmparin~ probes for isolallon of
`human-cONA clones (below),-The arrows under each Insert line
`Indicate Individual sequencing reactions.
`Figure 2 shows TG transfer 3divlty In normal S\lbjects.
`30 Protoin-stlmulated transfer of "C-TG from donor SUV to acceptor
`SUV was measured In homogenized Intestinal biopsies obtained
`· from five normal subjects. The rasulls are expressed as the
`percentage of donor TG transferred per hour as a function of
`· homogenized Intestinal biopsy protein.
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`Figure 3 shows TG. transfer activity in abelalipoproleinemlc
`subjects. Prolein·stimulated lransfer of 1•c-TG from donor SUV·to
`acceptor SUV was measured In homogenized Intestinal biopsies
`obtained from four abetallp0protelnemlc subjects. The results are
`expressed as lhe percentage of donor TG transferred'hour as a
`function or homogenized intestinal biopsy protein.
`Figure 4 shows TG transfer activity In control subjects.
`Protein stimulated transfer of '4C-TG lrom ·donor SUV 10 acceptor
`SUV In homogenized Intestinal biopsies were obtained from three
`control subjects, one with chylomlcron retention disease (open
`circles), one with homozygous hypobetallpoprotelnemia (softd
`circles), and one non-fasted (x). Tho results are expressed as lhe
`percentage of donor TG transferred/hour as a function of
`homogenized Intestinal biopsy prote!n.
`Agurs 5 shows westem blot analysis of MTP In normal
`subjects. An alquot of purtlled bovine MTP (lane 1) or the post
`103,000 x g proteins following deoxycholale treatment or 23 pg of
`homogenized lntestlnal biopsies from 3 normal subjects (lanes 2-
`4) were fractionated by SOS-PAGE and lhen transferred lo
`nitrocellulose. ·The blots were probed with antl-88 kOa.
`Figure 6 shows western blot analysis of MTP in control
`subjects. An atiquot a1 purified bovine MTP (lane 1) or lhe po$t
`103,000 x g proteins following diJc.xycholate treatment of 15 µg, 25
`µg, and 25 J.19 homogenized intestinal biopsies from a subject with
`chylomicron retention disease (lane 2), a subject wi1h
`homo::ygous hypobetalipoproteinemia (lane 3), and a non-fasted
`·- subject-(lane-4);-respectively, were-fractionated by SOS-PAGE
`and then transferred to nHrocellulose. The blots were probed with
`anli-88 kOa.
`Figure 7 shows western blot anPlysls of MTP In normal
`subjects with affinily-purifled antibodies. An aliquol or purified
`bovine MTP (lane 1) or the post 103,000 x g proteins f~llowing
`deoxycholate treatment of 34 µg (lane 2) or 25 µg (lane 3) of .
`homogenized Intestinal biopsies from 2 normal subjects were
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`fractionated by SDS·PAGE and then transferred to nitrocellulose.
`The blots went probed with affinity purified anti-88 kOa.
`Figure 8 shows western blot.analysls or MTP in
`abetaHpoprotelnemlc subjects. An aliquot or purified bovine MTP
`(lane 1) or post 1 03,000 x.g proteins following deoxycholate
`treatment or 18 µg (lane 2), 23 µg (lane 3}, 23 µg (lane 4}, 23 µg
`(lane 5) or homogenized Intestinal biopsies from lour difterenl
`abetalipoproteinemic subjects went fractionated by SDS·PAGE
`and then transferred to nitrocellulose. In lanes 6 and 7, 100 pg or
`the whole Intestinal homogenate (subjects corresponding In lane
`4 and 5) was fractionated by SDS·PAGE and transferred to
`nitrocellulose. The blots were probed with antl-88 kOa.
`Agure 9 shows a South.em biol analysls of a gene defect In
`an abelallpoproleinemlc subject. Ten µg of genomic ONA from a
`control, the abetaDpoprotelnemlc subject (proband), and from the
`subject's mothor and father were cut to completion with Taq I,
`electrophoresed on 1 % agarose and transferred to nitrocellulose.
`Southern hybridization We$ performed using oxon 13 cDNA as a
`probe. Two hybridizing bands In the normal lane Indicated the
`presence of a Taq I site In the normal exon 13. One hybridizing
`band io the abetallpopmtelnemic subjeci lane demonstrated the
`absence of this restriction sequence In both alleles in axon 13,
`confirming a homozygous mutation In this subject. The
`heterozygous state In· the mother ond lather is shown by the three
`hybridizing bands, corresponding lo bolh the normal and the
`·mutant 1'9stridlon-patt_ems.
`-
`-- -- --
`Figure 10 shows -Inhibition In MTP-ealalyzed transport ol TG
`from donor SUV to acceptor SUV by compound A described
`hereinafter. Compound A was dissolved In DMSO and then
`diluted into 15140 buffer. Aliquots were added to a lipid trans.er
`assay to bring the compound to the Indicated final concentrations.
`DtASO ooncentrat!on In lhe assay never exceeded 2 µU600 µL. a
`concentration that was Independently determined to have minimal
`effect on the assay. MTP-catalyzed &pid transport was measured
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`for 30 minutes al 37°C. TG transfer was calculated and compared
`to a control assay without Inhibitor. Three Independent assay
`conditions were used to demonstrate MTP Inhibition by compound
`A. Assay conditions were: 8 nmol donor PC, 48 nmol acceptor PC,
`and 75 ng MTP (open circles); 24 nmol donor PC. 144 nmol
`acceptor PC, and 100 ng MTP (solld circles); 72 nmol donor PC, ·
`432 nmol acceptor PC, and 125 ng MTP (open squares).
`Figure 11 shows the dose response of Compound A on
`ApoB, ApoAI and HSA secretion from HepG2 cells. HepG2 cells
`1 O were treated with compound A at the Indicated doses for 16 hours.
`The concentration In the cell cunure media of apoB, apoAi and
`HSA after lhe incubation period was measured whh the
`appropriate ELISA assay and normalized to total cell protein. The
`data shown are expressed as a percentage of the control (OMSO
`only).
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`Figure 12 shows the effect of compound A on TG secretion
`from HepG2 cells. HepG2 cens wera treated with Cl)ITlpound A al
`the indicated dOS41S for 18 hours, the last two hours of which were
`in the presence of 5 µCVmL 3H-glycerol. The concentration of
`radiolabelled triglycerides In the cell cutture media was measured
`by qJantltative extraction, followed by thin layer chromatography
`analysis and norma6zation lo total cell protein. The data shown
`aro expressed as a percentage of the control (DMSO only).
`Figure 13 shows lnhlbHlon in MTP-catalyzed.transport of TG
`from donor SUV to acceptor SUV by compound B described
`-- -hereinafter. Compound B was dissolved In OMSO and then
`diluted into t 5140 buffer. Aliquots were added to a Upid .transfer
`assay to bring the compound to. the Indicated final concentrations.
`DMSO concentration in the assay never exceeded 2 µU600 µL, a
`concentration that was Independently determined to have minimal
`effect on the assay. MTP-catalyzed lipid transport was measured
`tor 30 minutes at 37"C. TG transfer was calculated and compared
`lo a control assay without !nhlbltor. Two ln~ependent assay
`conditions were used to demonst~te MTP inhibition by compound
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`B. Assay condlllons were: 24 nmol donor PC. 144 nmol acceptor
`PC, and 100 ng MTP (open drclas); 72 nmol donor PC, 432 nmol
`acceptor PC, and 125 ng MTP (solid drclas).
`Figura 14 shows the dose response of compound Bon
`5 ApoB, ApoAI and HS~ sacretlo" from HepG2 call~. HepG2 calls
`ware ireated with compound Bat the Indicated doses tor 16 hours.
`The concentration in the cell culture media of apoB, apoAI and
`HSA after the Incubation period was measured with the
`approprtata ELISA assay and normalized to total cell protein. The
`1 o data shown are expresSC'd as a perce'ntaga of the control (OMSO
`only).
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`DmlJ1)od Descrlptlgn pf the lnyentlgn
`Definition of terms
`The lollowing delif'itlons apply to the terms as used
`throughout this spacllicatlon, untass ~herwisa limllad in specific
`instances.
`The term ·MrP· refers to a polypeptide or protein complex
`that (1) If obtained from an organism (e. g., cows, humans, Jlle.),
`can be isolated from the microsomal fraction of homogenized
`tissue; and (2) stimulates the 1ransport ol trtgly-:eridas, cholesterol
`asters, or phospholipids from synthetic phospholipid vttsicles,
`membranes or lipoprotelns 10 synthetic vesicles, membranes, or
`lipoprotelns and which is distinct from the cholesterol aster transfer
`protein (Drayna JlLal.. lia.Wm JZZ, 632·634 (1987)) which may
`have similar cat2lytic properties. However, the MTP molecules of
`-the prosenflnvention do not nacassarlly need to be catatytlcally
`active. For ttxample, catalytically inactive MTP or fragments
`thereof may be useful In ralslr.g ar.tibodlas to the protein.
`The term •modilied8, when ralentng to a nucleotide or
`polypeptide sequence, means a nucleotide or polypeptide
`sequence which differs from 1he ;Mid-type sequence lound in
`nature.
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`Tha torm ~re1111ta1", when refen1r.; to a nucleotide
`sequence, means a nucleic acid sequence which is able to
`hybridize to an oligonucleotide pri::-ie based on the nucleotide
`sequence of lhe high molecular weight subunit of MTP.
`The p~rase •control regions• refers lo nucleoti1e sequences
`that regulate expression of MTP or any subunit thereof. including
`but not limited !o any promoter, silencer, enhancer element~.
`splice sites, transcnptlonal Initiation elements, 1ranscriptlonal
`~erminatlon elements, polyadenylation signals, translalional
`control elements, translational star1 site. translational termination
`site, and message stability elements. Such control regions may
`be located In sequences 5' or 3' to the coding region or In introns
`Interrupting the coding region.
`The phrase "stablllzlng· atherosclerosis as used in the
`present application refers to slowing down lhe development of
`and/or Inhibiting lhe formation of new atherosclero11c lesions.
`The phrase "causing the regression of" atherosclerosis as
`used in the present application refers 10 reducing And/or
`ellmlnatlng atherosclerotlc lesions.
`
`Use end utlllty
`The nccleic acids of the present invention can be used in a
`variety of ways In accordance with the present invention. For
`example, they can be used as DNA probes to scree.n other cONA
`and genomic DNA libraries so as to select by hybridization other
`ONA sequences that code for proleins related to the high
`molecular weight subunit of MTP. In adcfition, the nucleic acids of
`thA present i:wention coding for all or pan of the high molecular
`weight subunit of human or bovine MTP can be used as DNA
`probes lo screen other cDNA and genomic ONA libraries to select
`by hybridization DNA seqUflnces that code for MTP molacules
`from other crganlsms. The nucleic acids may also be used lo
`generate primers to amplify cDNA or genomic DNA using .
`polymerase chain reaction (PCR) techniques. The ONA
`
`13 of 120
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`
`2091102
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`• 12.
`
`DC21a
`
`5
`
`15
`
`sequences of the present Invention· can also be used to Identity
`adjacent sequences i:l the cDNA or oenome; for example, those
`that encode the gene, its flanking sequences and its regulatory
`elements.
`· The polypeptides of the present Invention are useful In the •
`study of tho charaderistics of MTP; for example, Its structure,
`mechanism of acUon, and role In Dpld metabolism or lipoproteln
`particle assembly.
`·
`Various other methods of using the nucleic acids,
`1 O polypeptides, expression vectors and host cells are described In
`detail below.
`In carrying out the meth:>ds of the present Invention, the
`agents that decrease the activity or amount of MTP can be
`administered to various mammalian species, such as monkeys,
`dogs, cats, rats, humans, me., In need of such treatment. These
`agents can be administered systemlcally, such as orally or
`parenterally.
`The agent!; thal docraase the activity or amount of MTP can
`be Incorporated In a conventional systemic dosage form, such as
`a tablet, capsule, ellxlr or Injectable 'lo1TI1ulatlon. The above
`dosage forms wlll also Include the necessary physlol09lcally
`acceptable carrier material, exclplent, hJbrlcant, buffer,
`antibacterial, bulking agent (such as mannltol), anti-oxidants
`(ascorbic acid or sodium bisutllte) or the tike. Oral dosage forms
`are preferred, although parenteral forms are quite satisfadory as
`well.
`
`20
`
`25
`
`The dose admlnlmered must be carefully adjusted
`according to the age, weight, and condition or the patient, as well
`as the route of administration, dosage form and regimen, and the
`desired result. In general, the dosage forms described above may
`be administered In single or divided doses of one to four times
`dally.
`
`30
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`14 of 120
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`2091102
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`-13-
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`DC21a
`
`5
`
`Delalled description or specific embodiments
`Nuc!elc adds
`The present Invention concerns an Isolated nucleic acid
`moleeule comprisln'1 a nucleic add sequence coding for all or part
`of the high molecular weight subunit of MTP. Praferably, the
`nucleic acid molecule Is a DNA molecule and the nucleic acid
`sequence Is a ONA sequence. Further praferred Is a nuclefo acid
`sequence having the nucleotide sequence as shown In SEO. ID.•
`NOS. 1, 2, 5, 7, 8, 1 together with 5, 2 together with 7, the first 108
`baseJ of 2 together with B, or the first 108 bases of 2 together with
`7 and 8 or any part theraof, or a nucleic acid sequence
`complementary to one of th9sa ONA sequences. In the case of a
`nucleotide sequence (e.g., a DNA sequence) coding for part of the
`high moler.ular weight subunit of MTP, It Is pl'8ferred that the
`nucleotide r.equence be at least about 15 sequential nucleotides
`In length, mora preferably at least about 20 to 30 soquentlal
`nucleolljes In length.
`The foDowlng text shows a bovine cDNA nucleotide
`sequence (SEO. ID. NO. 1), a human eDNA sequence (SEO. ID .
`.20 NO. 2), a comparison of the human and bovine eDNA sequences,
`the bovine amino add sequence(SEO. ID. NO. 3), the human
`amino acid sequence (SEQ. ID. NO. 4), and a comparison of the
`human and bovine amino acid sequences. In the sequence
`comparisons, boxed regions represent perfoct Identity between
`the two sequences.
`
`1 O
`
`15
`
`25
`
`15 of 120
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`

`
`2091102
`
`-14-
`
`OC21a
`
`BOVINE cDNA SEQUENCE
`
`(SEQ. ID. NO. l)
`
`~T ACI'CCACI'GA >Lil 11 IICIC ~lCW•GCA ~ !':CJ
`
`~ G1G3Xl'ACC GAATITCATC ~T GICGCIIIAC
`
`100
`
`16 of 120
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`

`
`2091102
`
`-15-
`
`DC21a
`
`BOVINE cDNA SEQUENCE
`
`(SEQ. ID. NO. 1, continued)
`
`50
`40
`30
`20
`10
`1 23456i890 1234567890 1234567890 1234567890 1234567890
`
`~ 'l'CCTO:rn. MLIIICIC ™1Glu CMI IGXIC
`
`1100
`
`CTmTC.m .MGI IGIGI'CA ~ TOCANCrGA ~AAT 1250
`
`CATGA.b."1\J1N .. CIGICLGIAC ~ GXATCATlT ~ 1550
`
`~T GAATNGOC .:;:m:IC.CCA ~ c;.:rccrm
`
`:!.65C
`
`17 of 120
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`

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`2091102
`
`-16-
`
`OC21a
`
`BOVINE cONA SEQUENCE
`
`(SEO. ID. NO. 1, continued)
`
`:J
`50
`40
`30
`20
`·4•9~E/?CQ '2'4567890 '2'456/890 123d567890 ~?34567890
`AIIC .. !.~ CIQ;IIC'!a:i CA'ITCTAAG.'.i ~ 'ImC.ATCTI'
`
`1850
`
`.~ CCTATOCI'GG CTIG10G:T CICCICI llG ATG'rrn
`
`2000
`
`tCCCG:ro:A ~ 'rn\CATGT ~ GIIIO:IO::I
`
`2550
`
`OJICCJ>m GCAGT'I'CCAG IGGI IGGI I l TGAAAC1GU G:J:a.. .1.Gl I
`
`2600
`
`I I'OCICICTG mDGr G!Tl7CATAT TI:ACX:TGrAT TrNGGTIT
`
`2700
`
`18 of 120
`
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`

`
`2091102
`
`-17-
`
`OC21a
`
`BOVINE cDNA SEQUENCE
`
`ISEQ. ID. NO. l, continued)
`
`:o
`50
`40
`30
`20
`:?<4~67800 1?~4567890 ~?<4567890 1234567890 1234561890
`
`19 of 120
`
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`

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`2091102
`
`-18-
`
`OC21a
`
`HUMAN cONA SEQUENCE
`(SEQ. IO. NO. 21
`
`50
`40
`30
`20
`10
`1234567890 1234567890 1234567890 1234567890 1234567890
`
`AIICI ICIIG CIGIG:llll 'ICICIG:IIC AlllCCTCAT ATl'ClG:'ITC
`
`100
`
`CPCCTATAPG AT'NJNIC:JCA OCI l IGI IAT AG:IGIO::l I ~ 750
`
`20 of 120
`
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`

`
`2091102
`
`-19-
`
`DC21a
`
`HUMANcDNASEOUENCE
`ISEQ. ID. NO. 2, continued)
`
`50
`40
`30
`20
`10
`1234561890 1234561§90 '.2:{;i61B90 1234561890 1234561690
`CAACCITTCC ~ ClGl'CAG?\AA CllCCI03CC TTCA'I'TCAG:
`
`1050
`
`OCN::CAT .... »T ~ ALLIIICICI Am:::croro:; AillC:CIICT
`
`1250
`
`'I'QIG'.;'fGAAG AAGNJ:ITAA N:!GV>.TATA CCN::C»NiC CGrAAAGITC
`
`1650
`
`a:::I'CN'...AATT A~~ a:;A:ICI !Cll c1c:ccr;.,a;c
`
`1900
`
`21 of 120
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`2091102
`
`-20-
`
`DC21a
`
`HUMAN cDNA SEQUENCE
`(SEQ. ID. NO. 2, continuedl:
`
`40
`50
`10
`30
`20
`12345618QO 1234567890 4234567890 1234567890 1234567890
`
`T1CI'TGAAT1' IGIC1ux::a:; ~ ~T GN:rNCfAC
`
`2eoo
`
`1 IG:ICICIG ~ GITTPCATAT TI1\CCIGTAT 'IT.AAGA1TIT
`
`2850
`
`TGrAA.r..AN:r. T/JCAAAANC ~ 'l'CAAA'ITI'G:i GTATATCi:x;.
`
`2900
`
`GI ICI l:llLTI TFCI'TATKJ: 'I'C'ICOCAMT CTCAll IG:il ~ 3000
`
`22 of 120
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`

`
`2091102
`
`-21-
`
`OC21a
`
`HUMAN cONA SECUENCE
`ISEQ. ID. NO. 2, continued)
`
`50
`40
`30
`20
`10
`1234567890 1234567890 1234567890 1234567890 1234567990
`
`~ ~ C'C"l1';1 I I !GI TICAACAATT Tl'TG1\TCAAT
`
`3100
`
`23 of 120
`
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`2091102
`
`-22-
`
`OCZ1a
`
`BOVINEttiUMAN cDNA SEQUENCE COMPARISON
`
`:sc
`
`JO!
`00
`
`T1'C.1AA.\GTCOACCTACCAGC;.;TCATCMCACAA
`~r~r~M:GC:TCATCAACACAAA
`
`·Uf.
`.;:o
`
`'f<t;rc~~~~~
`'t9'C~~AA ~~}j
`
`·~=
`
`CAT=t:CCATrGr=M;GTCTT
`IXAT=A~:rc=M:CTCT
`
`'f:'CtCTC
`CTrcrcTC
`
`c:cATTACTUCTtCllAAC;Cr?C
`
`AT
`
`; )! :
`: :oo
`
`:i:n
`!JlC
`
`30\llHI:
`llllMAll
`
`aov:11E
`HUMAN
`
`80VINE
`"llHAll
`
`IOVl!f1:
`MUMAN
`
`BOVINE
`llUKAll
`
`a:iv:~.E
`~ lift;MAN
`
`90VlNE
`i-IUMAN
`
`t1ov:~c
`ilUKAN
`
`BOVINI
`~UIWO
`
`l'OVIMt
`.. U!l\MI
`
`80VINE
`ftUKAtl
`
`24 of 120
`
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`

`
`2091102
`
`-23-
`
`DC21a
`
`BOVINflHUMAN cONA SEOUENCE COMPARlsoN
`
`!Ul
`tlCO
`
`:·121
`; t ,,
`
`1HI
`!400
`
`1401
`:sso
`
`:rc:TTT~" TCTTC.\GCTCACA.. "CT J'C"
`ICTTTCa~CAC::TCM:A=:TCTC"
`
`l~Tt:l'CJ'(J.;1~1
`~MJ'flTTC1':&GT1'C\Xl\
`
`·1.:\·:sr
`·.~Ji:
`
`Si:J\•;:o.1t
`"ta M.AS
`
`i::>v:~r.
`.... M!-
`
`30\';~[
`wi~M.it,;
`
`•\;!,_·: .. t
`•r.:tU.r•
`
`~:J·1:•:t
`··"..,.~!I
`
`:-.-:·11'"'·
`.. ·.:!W.\1"
`
`'!'.'lV:Nt.
`-1:.iP"~f;
`
`. .a:ov:111t
`tl'!l'!AN
`
`25 of 120
`
`PENN EX. 2167
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`IPR2015-01836
`
`

`
`2091102
`-24-
`
`DC21a
`
`DOVINEMJMAN cONA SEOJENCE COMPARISON
`
`aov:~r.
`11\l!'ltA:W
`
`l!OVJNE
`.. u .......
`
`BOVINE
`HllMMI
`
`sov:•11:
`HllMMI
`
`~~-----·---·-------·--
`t
`,
`•
`
`~ANJtCCACACAAGCACW
`
`ltO~
`lOTD
`
`114'
`lt•~
`
`ltCQ
`nu
`
`26 of 120
`
`PENN EX. 2167
`CFAD V. UPENN
`IPR2015-01836
`
`

`
`2091102
`
`-25-
`
`DC21a
`
`BOVINE PROTEJN SEQUENCE
`(SEQ. 10. NO. 31
`
`w
`~
`~
`~
`-~
`.:.2,456"'ii!QQ i231561890 , "34567890 1234567890 ~ 2345678 :IQ
`KLTISTEVFL OFGO«.OJS 'v"G'iR:iSSNVD ~ ~UQI1M :0
`
`IR~ SilG<IVSPO KLEI..KTTFAS VRll<PGl<OVA AIIKAVDSKY
`
`TA.IP NG:}IF QSK.Cl<G:PSL SEHWJS !lOOi I.QPDNLSK1'E AVRSFLAFU<
`
`: iIBTAKKEEI LQUJQ\ENi<E VtI?QLVOAvr SAQIPOSWA IIDFIDF'KST
`
`29)
`
`JOU
`
`:;so
`
`ILYSGSGILR PSNINIFQYI EKI'PUiGIQJ VIE10:;tEAL IAATPDEXll ·
`
`650
`
`27 of 120
`
`PENN EX. 2167
`CFAD V. UPENN
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`
`

`
`2091102
`
`.:.26-
`
`OC21a
`
`HUMAN PROTEIN SEOUENCE
`(SEQ. ID. NO. 4)
`
`50
`40
`30
`10
`'~
`.12~~~6?89