Vol.
`
`4,
`
`993-1004,
`
`April
`
`1998
`
`Clinical
`
`Cancer
`
`Research
`
`993
`
`Recombinant
`Interactions
`Activity
`
`Heregulin-Pseudomonas
`with
`the Heregulin
`in Vivo1
`
`Fusion
`Exotoxin
`Receptors
`and Antitumor
`
`Proteins:
`
`injection
`intratumor
`by
`achieved
`was
`regression
`Continuous
`5 days.
`for
`day
`proteins
`fusion
`per
`of HRGfH-PE38KDEL
`of EGF-like
`domain
`istration
`at
`a dose
`of
`40
`a miniosmotic
`pump
`via
`days
`receptor
`positive
`the
`growth
`of ErbB-4
`inhibited
`lines
`in athymic
`receptor
`negative
`cell
`ErbB-4
`We
`conclude
`that
`there
`therapeutic
`potential
`is
`toxins
`in the
`therapy
`cancers
`overexpressing
`or ErbB-2
`plus
`ErbB-3
`receptors.
`
`of
`
`of
`
`7
`
`of
`i.p.
`
`1 (cid:1)tg
`admin-
`for
`(cid:1)tg/kg/day
`but
`
`not
`mice.
`nude
`of HRG-PE
`the ErbB-4
`
`Dajun
`
`Yang,2
`
`Chien-Tsun
`
`Kuan,
`
`Jeniffer
`
`Payne,
`
`Ako
`
`Kihara,
`
`Ann
`
`Murray,
`
`Ling-Mei
`
`Wang,
`
`Maurizio
`
`Alimandi,
`
`Jacalyn
`
`H.
`
`Pierce,
`
`Ira
`
`Pastan,
`
`and
`
`Marc
`
`E.
`
`Lippman
`
`Lombardi
`Departments
`Medicine
`Medical
`Molecular
`Biology
`Bethesda,
`
`and
`J. P., A. M., M. E. L.]
`[D. Y.,
`Center
`Cancer
`[D. Y.]
`and Molecular
`Biology
`of Biochemistry
`University
`[M. E. LI,
`Georgetown
`Pharmacology
`and
`DC
`20007;
`and
`Laboratories
`of
`Washington,
`Center,
`[C-T. K., A. K.,
`I. P.] and Cellular
`and Molecular
`Biology
`[L-M. W., M. A.,
`J. H. P.], National
`Cancer
`Institute,
`NIH,
`Maryland
`20892
`
`and
`
`factor
`of
`
`of
`
`in-
`
`are
`
`INTRODUCTION
`
`ErbB-2/neu
`and
`EGFR3
`transmembrane
`related
`structurally
`development
`a role
`in the
`that
`play
`family,
`2). Members
`of
`this
`genes,
`erbB-3
`and
`erbB-4
`lines,
`human
`cancer
`cell
`binding
`domains
`of ErbB-3
`homology
`to each
`other
`of
`known
`members
`the
`studies
`munostaining
`(10 -12),
`breast
`cancer
`(14).
`The
`expression
`
`of
`
`family
`a
`for
`prototypes
`receptors)
`I
`(type
`proteins
`of cancer
`and
`progression
`identified
`including
`the
`recently
`in
`various
`often
`overexpressed
`are
`The
`ligand-
`as
`breast
`(3-9).
`such
`ErbB-4
`share
`significantly
`more
`and
`other
`than
`they
`do
`to the
`two
`(-65%)
`Recent
`family
`(41-46%).
`im-
`receptor
`ErbB-3
`is
`overexpressed
`in
`show
`that
`ovarian
`cancer
`( 13)
`and
`prostate
`ErbB-4
`is
`elevated
`in
`breast
`cancer
`
`of
`
`(1,
`
`cancer
`cell
`
`but
`
`of
`
`fact
`
`ABSTRACT
`receptors
`Growth
`targeted
`for development
`tyrosine
`the
`type
`I receptor
`(EGF)
`mal
`growth
`factor
`are
`often
`overexpressed
`cluding
`breast.
`Recently,
`and
`ErbB-4
`receptors
`are
`entiation
`Eight
`factor.
`tracellular
`EGF-like
`and
`and
`isoforms
`truncated
`constructed.
`were
`The
`to the
`native
`ilar
`HRG
`rylation.
`The
`EGF-like
`fused
`to PE38KDEL
`with
`a IC50
`of
`(cid:17)0.001
`to PE38KDEL
`were
`The
`HRG-Pseudomonas
`tremely
`high
`activity
`tor,
`alone
`together
`or
`receptor
`Cells
`family.
`press
`receptor,
`ErbB-3
`exhibited
`moderate
`toxins
`have
`little
`EGFR,
`ErbB-2,
`
`opportunities
`unique
`provide
`Members
`therapy.
`anticancer
`family,
`including
`epider-
`kinase
`(EGFR)
`and
`ErbB-2/neu,
`receptor
`human
`cancer
`cells,
`in various
`shown
`that
`both
`ErbB-3
`it has
`been
`for
`heregulin
`(HRG)INeu
`differ-
`of
`chimeric
`toxins
`composed
`the
`ex-
`domains
`of
`four
`different
`HRG
`exotoxin
`(PE38KDEL)
`Pseudomonas
`sim-
`fusion
`proteins
`exhibited
`activity
`in inducing
`ErbB
`receptors
`phospho-
`of HRG(cid:1)fl
`and
`HRGI32
`domain
`showed
`highest
`cytotoxic
`activity,
`the
`nglml.
`a isoforms
`that were
`fused
`The
`100-fold
`active
`than
`the
`isoforms.
`less
`ex-
`(PE)
`toxins
`show
`exotoxin
`recep-
`expressing
`ErbB-4
`cells
`against
`ErbB
`members
`of
`the
`other
`with
`but
`ex-
`express
`ErbB-4
`not
`do
`that
`or EGFR,
`the ErbB-2
`with
`together
`HRG-PE
`to HRG-PE
`toxins.
`sensitivity
`expressing
`or
`no
`activity
`against
`cells
`or ErbB-3
`alone. More
`an 80% tumor
`
`than
`
`1/16/98.
`accepted
`12/12/97:
`defrayed
`article
`were
`of
`this
`This
`article must
`therefore
`with
`18 U.S.C.
`Section
`
`the
`by
`part
`in
`be hereby marked
`1734
`solely
`
`to
`
`revised
`2/17/97;
`Received
`of
`publication
`The
`costs
`of page
`charges.
`payment
`in accordance
`advertisement
`fact.
`this
`indicate
`part
`Jennie
`from
`funding
`by
`in
`supported
`was
`work
`I This
`(Specialized
`SPORE
`Institute
`Cancer
`and National
`Foundation
`1P50CA58
`Cancer
`(NIH
`in Breast
`grams
`of Research
`Excellence)
`in part
`were
`supported
`The
`nude mice
`experiments
`in
`vivo
`1008).
`Facility
`(CA5
`Lombardi
`Cancer
`Center
`Animal
`Core
`2 To whom
`requests
`for
`reprints
`should
`be
`addressed,
`Cancer
`Center,
`Georgetown
`University
`Medical
`Center,
`DC 20007.
`
`Zoline
`Pro-
`185).
`the
`
`by
`
`at Lombardi
`Washington,
`
`correlations
`clinical
`(9),
`yet
`not
`are
`The
`available.
`lines
`tumor
`cells
`breast
`many
`preferentially
`co-overexpress
`that
`with
`ErbB-3
`or EGFR
`and/or
`ErbB-4
`(3-9)
`receptors
`ErbB-2
`the
`importance
`of
`all
`these
`receptors
`in
`the
`pathogen-
`supports
`breast
`cancer.
`The
`extracellular
`accessibility
`of
`these
`esis
`makes
`them
`attractive
`potential
`targets
`specific
`proteins
`therapy.
`antitumor
`isolate
`Attempts
`to
`discovery
`factor
`
`of
`
`a
`
`for
`a ligand
`named
`alternatively
`
`ErbB-2
`
`have
`various
`
`led
`
`the
`
`to
`groups,
`
`by
`
`for
`
`e.g.,
`
`gp3O
`
`(15),
`
`HRG
`
`(16),
`
`NDF
`
`(17),
`
`acetylcholine
`
`receptor
`
`factor
`
`of
`EGF-like
`
`inducing
`are
`products
`domain.
`phosphorylation
`of HRG
`contains
`the EGF
`family
`to EGFR
`(16-17,
`that
`both
`ErbB-3
`presence
`of
`one
`
`(18),
`
`and
`
`glial
`
`growth
`
`factor
`
`(19),
`
`all
`
`of which
`
`the
`
`same
`domain
`
`gene
`is
`
`contain
`
`EGF-like
`single
`a
`and
`stimulation
`the
`for
`sufficient
`EGF-like
`domain
`The
`receptor(s).
`of
`p185erbB
`characteristic
`that
`are
`residues
`six
`cysteine
`of growth
`factors;
`however,
`HRG
`does
`not
`20-22).
`More
`recently,
`it has
`been
`discovered
`and ErbB-4
`are
`receptors
`for HRG
`and
`that
`of
`these
`two
`receptors
`is
`required
`for
`
`of
`
`of
`bind
`
`the
`the
`
`factor
`growth
`epidermal
`are: EGFR,
`used
`abbreviations
`3 The
`growth
`epidermal
`EGF,
`Neu
`NDF,
`heregulin;
`factor:
`HRG,
`transforming
`exotoxin;
`TGF,
`factor;
`PE,
`tion
`Pseudomonas
`serum;
`improved
`MEM;
`FBS,
`fetal
`bovine
`IMEM,
`factor;
`of HRG;
`(2,3-bis[2-methoxy-4-nitro-S-sulfophenyl]-
`domain
`like
`2H-tetrazolium-S-carboxanilide
`inner
`salt;
`Ab,
`antibody;
`clonal
`antibody:
`HSA,
`serum
`albumin.
`
`receptor;
`differentia-
`growth
`EGF-
`
`HEL,
`
`mAb,
`
`mono-
`
`Xli’,
`
`human
`
`
`
`clincancerres.aacrjournals.org Downloaded from
`
`on December 31, 2014. © 1998 American Association for Cancer
`Research.
`
`IMMUNOGEN 2158, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`(cid:1)
`

`

`994
`
`Recombinant
`
`Heregulin-Pseudomonas
`
`Exotoxin
`
`through
`by HRG
`are
`and/or
`ErbB-4
`bind
`to HRG/NDF,
`when
`it
`is expressed
`both
`ErbB-2
`and
`affinity
`compared
`27).
`ErbB-2
`appears
`signaling
`through
`and/or
`ErbB-3
`(22,
`identified
`
`(21-
`heterodimerization
`only
`the
`as
`expressed
`does
`ErbB-2
`whereas
`28).
`In
`(22,
`alone
`HRG/
`bind
`ErbB-3
`expressing
`those
`to
`partic-
`to indirectly
`receptor
`heterodimer-
`24,
`29,
`30). No
`ligand
`bind
`directly
`to
`the
`
`can
`
`that
`
`24,
`
`cell
`nents
`tural
`sible
`
`toxin
`by
`
`inactivates
`irreversibly
`that
`catalytically
`modifying
`key
`of
`machinery
`is made
`three
`Ia
`(amino
`acids
`1-252)
`is
`middle
`domain,
`domain
`II
`
`and
`
`of ErbB-2
`activation
`ErbB-3
`28). When
`they
`receptor
`in cells,
`not
`bind
`to HRGINDF
`addition,
`cells
`expressing
`NDF
`increased
`with
`ErbB-3
`(22,
`alone
`ipate
`in HRG-mediated
`ization
`with
`ErbB-4
`been
`has
`conclusively
`ErbB-2
`receptor.
`is
`a bacterial
`PE
`protein
`synthesis
`of
`the
`translational
`domains
`(31).
`Domain
`for
`cell
`binding;
`the
`
`target
`compo-
`struc-
`respon-
`(amino
`(amino
`factor
`elongation
`of
`ADP-ribosylation
`catalyzes
`400-613)
`acids
`func-
`no
`known
`has
`acids
`365-399)
`lb
`(amino
`2 (3 1). Domain
`loss
`of
`without
`deleted
`lb
`can
`be
`of
`domain
`tion,
`and most
`of amino
`acids
`252-613
`of PE, with
`activity
`(31).
`PE38
`consists
`365-380.
`Modification
`the COOH
`a deletion
`of
`amino
`acids
`Lys-Asp-Glu-Leu
`increases
`terminus
`by
`tetrapeptide
`(KDEL)
`toxin
`activity
`(32).
`A number
`of
`recombinant
`have
`been
`toxins
`developed
`fusing
`growth
`factors
`(ligand),
`single-chain
`anti-
`gen-binding
`proteins,
`and
`cytokines
`to PE.
`These
`chimeric
`im-
`munotoxins
`or oncotoxins
`are
`very
`cytotoxic
`to target
`cells
`and
`have
`potent
`tumor-inhibiting
`activity.
`Several
`clinical
`trials
`are
`underway
`(31).
`TGF-a-PE4O,
`which
`targets
`EGFR,
`has
`evaluated
`in a Phase
`I trial
`of bladder
`carcino-
`mas
`(33).
`and
`of HRG
`composed
`toxins,
`Chimeric
`as a novel
`therapeutic
`agent
`a potential
`have
`We
`have
`previously
`described
`cancer.
`breast
`recombinant
`HRG-PE
`fusion
`proteins,
`several
`shown
`to have
`cytotoxic
`activities
`been
`in
`vitro
`a
`series
`of
`studies
`of
`the
`cytotoxic
`efficacy
`report
`chirneric
`toxins
`for
`a panel
`of
`HRG-PE38KDEL
`in
`nude
`mice
`and
`correlation
`of HRG
`receptors
`and
`cytotoxic
`
`acids
`
`253-364),
`
`mediates
`
`translocation;
`
`and
`
`domain
`
`III
`
`by
`
`in
`
`vivo
`
`now
`been
`
`in
`
`cells
`specific
`
`and
`vitro
`expression
`
`of
`
`may
`PE38KDEL,
`of
`for
`the
`treatment
`of
`the
`construction
`have
`and
`they
`now
`(34).
`We
`various
`cancer
`the
`
`of
`breast
`between
`activity.
`
`AND
`Cell
`
`obtained
`MD).
`by
`
`by
`
`in
`
`METHODS
`MATERIALS
`were
`lines
`cell
`Most
`Culture.
`and
`Cells
`(Rockville,
`Collection
`Type
`Culture
`the American
`from
`kindly
`provided
`cancer
`cell
`line N87 was
`human
`gastric
`The
`Dr. R. King
`(Lombardi
`Cancer
`Center,
`Georgetown
`University;
`Ref.
`35). MDA-435/LCC6
`cells
`were
`generously
`provided
`Dr. R. Clarke
`(Lombardi
`Cancer
`Center,
`Georgetown
`Univer-
`sity;
`Ref.
`Cells
`routinely
`maintained
`IMEM
`36).
`were
`(Biofluids,
`Rockville,
`with
`10% FBS.
`32D
`and
`var-
`cells
`MD)
`ious
`ErbB
`receptor
`32D
`cells
`were
`as
`in a
`cultured
`transfected
`previous
`report
`(49).
`were
`maintained
`in a humidified
`Cultures
`CO2.
`incubator
`at 37#{176}Cand
`5%
`Toxins.
`the Chimeric
`of
`Construction
`and
`Purification
`described
`of HRG
`to the modified
`PE (PE38KDEL)
`was
`(34).
`Briefly,
`the
`four
`isoforms
`extracellular
`the
`of
`(HRG)
`and
`four
`isoforms
`(amino
`acids
`of HEL
`for HRGa1
`acids
`175-247
`for HRG(cid:1)3
`
`Fusion
`previously
`domains
`
`175-242
`
`the
`, amino
`
`1 , amino
`
`coli
`bodies.
`and
`
`acids
`
`175-239
`were
`HRG(cid:1)33)
`produced
`were
`Escherichia
`inclusion
`guanidine
`Q-Sepharose,
`cellular
`agarose
`lyze
`were
`
`and
`
`amino
`and
`PE38KDEL.
`promoter-based
`where
`each
`recombinant
`purified
`5/5,
`HRG
`
`and
`
`175-242
`
`for
`proteins
`system
`within
`
`in
`
`in
`of
`
`acids
`Recombinant
`expression
`accumulated
`were
`dissolved
`sequential
`use
`columns.
`Extra-
`by
`heparin-
`used
`to
`ana-
`proteins
`
`toxin
`proteins
`through
`TSK-250
`were
`purified
`SDS-PAGE
`was
`the
`final
`recombinant
`
`HRG(cid:1)32,
`for
`to the
`fused
`the T7
`with
`(BL21),
`The
`renatured,
`Q HR
`Mono
`native
`of
`domains
`of Q-Sepharose.
`instead
`fraction,
`column
`each
`least
`95% pure.
`at
`of Chimeric
`Activity
`Assay
`of Biological
`synthesis
`on protein
`of chimeric
`toxins
`effect
`of
`inhibition
`Assays
`previously
`(34).
`described
`to directly
`plastics
`out
`carried
`on
`ation
`were
`tetrazolium/formazan
`killing
`activity.
`A soluble
`plate
`was
`performed
`cell
`growth
`in a 96-well
`grown
`in IMEM
`with
`4000
`cells/well)
`were
`concentrations
`of
`treated
`with
`increasing
`4 days
`of culture,
`(0.001-10
`(cid:1)ig/ml).
`After
`at 1.53
`rng/ml)
`phenazine
`methosulphate
`and
`incubated
`for
`4 h at 37#{176}C.A450
`nm was
`the
`Dynatech
`Model
`MR700.
`To
`examine
`colonies,
`protein
`to penetrate
`through
`soft-agar
`with
`0.33%
`sion
`(10,000
`cells/ml)
`were
`mixed
`1% agarose.
`plated
`on
`of
`a bottom
`layer
`with
`top
`different
`concentrations
`of HEL(cid:1)l-PE38KDEL
`ml of
`culture
`medium
`were
`added
`to the
`for
`2 weeks.
`The
`number
`of
`colonies
`diameter
`and
`formed
`after
`the
`incubation
`
`was
`
`The
`as
`
`Toxins.
`determined
`cell
`prolifer-
`of
`measure
`the
`cell-
`(XTT)
`assay
`for
`(37).
`Cells
`(2000-
`10% FBS
`and were
`recombinant
`toxins
`XTF
`(1.0 mg/ml
`plus
`was
`added
`to each well
`
`measured
`ability
`cells
`of
`
`the
`with
`a
`fusion
`of
`in suspen-
`agarose
`and
`The
`next
`day,
`mixed
`with
`and
`incubated
`>60
`(cid:1)irn
`was
`counted
`
`in
`
`1
`
`top
`that
`
`layer
`were
`period
`
`X
`
`106)
`were
`buffer
`1%
`
`and
`de-
`IL).
`
`with
`IL).
`in-
`To
`treated
`
`with
`Inc.,
`Inc.,
`CA),
`Inc.),
`
`using
`
`1-2
`
`and
`X
`The
`and
`
`of
`were
`
`system.
`Analysis
`Image
`Lomb
`and
`a Bausch
`Receptors.
`of ErbB
`Blot
`Western
`(1-2
`Cells
`Cells
`10% FBS.
`dishes
`with
`100-mm
`into
`plated
`were
`lysis
`in 1 ml of
`and
`PBS
`lysed
`ice-cold
`with
`twice
`washed
`(pH 7.4),
`[50 mr(cid:1)i Tris-HC1
`5 msi MgCl2,
`150 mr(cid:1)i NaCl,
`1 msi
`phenylmeth-
`5 mss
`EGTA,
`5 msi
`EDTA,
`Triton
`X-100,
`p(cid:1)g/ml
`leupeptin,
`aprotinin,
`50
`50
`ylsulfonyl
`fluoride,
`jig/mi
`A protein
`concentration
`was
`2 ms(cid:1)
`sodium
`orthovanadate].
`acid method
`(Pierce,
`Rockford,
`termined
`by
`bicinchoninic
`subjected
`to
`8-20%
`SDS-PAGE
`were
`Thirty
`protein
`of
`(cid:1)ig
`transferred
`to nitrocellulose
`mem-
`CA)
`and
`(Novex,
`San Diego,
`Ab,
`detected
`a
`specific
`was
`brane.
`Each
`receptor
`e.g.,
`Placid,
`Biotechnology,
`Lake
`NY),
`mAb
`(Upstate
`EGFR
`Sciences
`Cambridge,
`MA),
`ErbB-2
`mAb
`(Oncogene
`Fremont,
`or
`antiphosphotyro-
`ErbB-3
`(NeoMarkers
`Inc.,
`mAb
`(Upstate
`Biotechnology,
`and
`visualized
`sine
`Arlington
`Heights,
`enhanced
`chemiluminescence
`(Amersham,
`Individual
`Receptor
`Tyrosine
`Phosphorylation.
`cells
`vestigate
`receptor
`activation
`by
`the
`fusion
`proteins,
`with
`or without
`HEL(cid:1)31-PE38KDEL
`were
`immunoprecipitated
`ErbB-3,
`with
`specific
`EGFR,
`ErbB-2,
`and
`ErbB-4
`antibodies
`Ab.
`with
`an
`separately
`then
`blotted
`antiphosphotyrosine
`with
`plated
`dishes
`Briefly,
`106 cells
`were
`into
`100-nun
`serum-
`were
`with
`10% FBS.
`next
`day,
`cells
`washed
`twice
`were
`5 h. Cells
`that
`free
`IMEM
`incubated
`in
`medium
`for
`then
`stimulated
`with
`10 nM of HEL(cid:1)3l-PE38KDEL
`for
`10 mm at
`37#{176}C.The
`cells
`were
`washed
`twice
`with
`ice-cold
`PBS
`and
`lysed
`in
`1 ml
`lysis
`buffer
`(same
`as
`above).
`Five
`hundred
`p.g
`of
`protein
`incubated
`with
`EGFR
`mAb
`(Upstate
`Biotechnol-
`
`Downloaded from
`
`on December 31, 2014. © 1998 American Association for Cancerclincancerres.aacrjournals.org
`
`
`Research.
`
`IMMUNOGEN 2158, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`Inc.),
`Cruz,
`
`Inc.),
`Ab
`
`ErbB-2
`(Santa
`with
`with
`at
`
`proteins
`ErbB-4
`
`and
`
`Science
`(Oncogene
`mAb
`ogy,
`Santa
`Biotechnology,
`Cruz
`ErbB-4
`rotation.
`Immunocomplexes
`gentle
`overnight
`A-Sepharose
`(CL-4B;
`protein
`incubated
`1 h
`and
`collected
`4#{176}Cfor
`NJ)
`taway,
`onto
`nitrocellulose
`electroblotted
`were
`Proteins
`Immunoreactive
`mAb.
`antiphosphotyrosine
`chemiluminescence
`ized
`using
`enhanced
`tion,
`the
`same
`immunoprecipitated
`the
`EGFR,
`ErbB-2,
`ErbB-3,
`tively.
`
`or
`or ErbB-3
`CA)
`at 4#{176}C
`were
`then
`Pisca-
`Pharmacia,
`by
`ultracentrifuge.
`and
`probed
`bands
`were
`(Amersham).
`were
`blotted
`antibodies,
`
`with
`visual-
`In addi-
`against
`respec-
`
`Animal
`nude mice
`the
`tail
`
`or
`
`female
`five
`four
`of
`through
`iv.
`injections
`of HEL(cid:1)1-
`amounts
`were
`moni-
`Animals
`2 weeks
`after
`for
`the
`doses
`that
`noting
`the
`animals
`were
`sacri-
`Tissues
`were
`examined
`
`Studies.
`Toxicity
`Groups
`single
`given
`were
`(NCr,
`nu/nu)
`of
`different
`(cid:1)i.l
`vein
`with
`100
`diluted
`in PBS-HSA
`(0.2%).
`PE38KDEL
`for
`weight
`loss
`and
`mortality
`tored
`daily
`The
`LD50
`was
`obtained
`by
`injection.
`50% death
`of
`animals.
`Some
`of
`caused
`and major
`organs
`were
`removed.
`ficed,
`by H&E-stained
`sections.
`evaluate
`To
`Pharmacokinetics.
`of
`pharmacokinetics
`the
`g)
`22-25
`female
`nude mice
`(weight,
`in blood,
`chimeric
`toxin
`the
`in 200
`1 (cid:1)i.g of HELI3
`1 -PE38KDEL
`of
`dose
`given
`a single
`were
`samples
`into
`the
`tail
`vein.
`Blood
`injection
`(cid:1)il
`of PBS-HSA
`by
`sinus
`or chest
`cavity
`at 2, 5,
`10, 20,
`orbital
`were
`drawn
`from the
`and
`300 mm after
`injection.
`Each
`240,
`30,
`45,
`60,
`120,
`180,
`and
`each
`time
`point
`represents
`the
`times,
`mouse
`was
`bled
`three
`three
`mice.
`The
`concentration
`of
`from
`mean
`of
`results
`obtained
`measured
`bioassay,
`in which
`was
`the
`recombinant
`toxin
`in
`PBS
`incubated
`with
`N87
`diluted
`serum
`samples
`were
`made
`purified
`HEL(cid:1)3l-
`curve
`cells.
`A
`standard
`parameters
`calculated
`using
`Pharmacokinetic
`PE38KDEL.
`program,
`(MicroMath
`Sci-
`curve
`fitting
`an exponential
`City,
`UT).
`Salt
`Lake
`entific
`Software,
`developed
`have
`In Vivo Antitumor
`Activity.
`We
`MDA-361/DYT2.
`termed
`of
`the MDA-36l
`cells
`line
`large
`tumors
`that
`grow
`derivative
`cell
`line
`forms
`no
`sively
`are
`highly
`vascularized,
`but
`there
`are
`and
`the
`in
`the
`expression
`of
`the
`ErbB
`receptors
`or
`sensitivity
`line
`cell
`(data
`not
`shown).
`Details
`of
`this
`HRG-PE
`vitro
`in
`of
`fusion
`will
`be
`elsewhere.
`Tail
`vein
`injection
`described
`was
`(LD10)
`of HEL(cid:1)31-PE38KDEL
`proteins
`p.g/kg
`50
`up
`to
`tumor
`injected
`day
`for
`8 days
`into mice
`bearing
`other
`every
`out
`xenograft.
`(intratumor)
`administration
`was
`carried
`Local
`into
`of
`by
`direct
`of
`the
`chimeric
`toxin
`the
`right
`side
`injection
`tumor
`at
`daily
`for
`1 p.g/tumor
`whereas
`of
`5 days,
`the
`left
`side
`with
`In
`tumor
`of
`mouse
`was
`treated
`PBS.
`other
`the
`same
`107,
`animals,
`pumps
`(model
`Corp.,
`Palo
`Alza
`miniosmotic
`used
`Alto,
`7 days)
`were
`to
`deliver
`fusion
`for
`0.5
`(cid:1)i1Th
`proteins
`implantation.
`Osmotic
`pumps
`containing
`through
`i.p.
`escalating
`doses
`of HRG
`chimeric
`toxins
`were
`placed
`through
`i.p.
`implantation,
`and
`the
`LD50
`for
`chimeric
`toxins
`using
`osmotic
`pumps
`was
`determined
`in
`pilot
`experiments.
`Minipumps
`were
`filled
`with
`100
`p.1 ofHEL(cid:1)3l-PE38KDEL
`PBS
`doses
`of
`or
`80
`(cid:1)i.gIkg/day
`for
`7
`days
`in
`sterile
`40
`106)
`containing
`0.2%
`HSA
`or with
`PBS
`alone.
`Cells
`(1-5
`the
`were
`injected
`day
`0
`into
`the
`mammary
`pad
`were
`female
`athymic
`nude
`mice,
`and
`filled
`osmotic
`pumps
`placed
`i.p.
`5 days
`after
`tumor
`implantation.
`Each
`treatment
`
`was
`
`by
`and
`using
`
`were
`RSTRIP
`
`a sub-
`This
`progres-
`changes
`to
`
`at
`
`X
`of
`
`fat
`
`CA;
`
`on
`
`Clinical
`
`Cancer
`
`Research
`
`995
`
`group
`lated
`
`consisted
`by
`using
`
`five
`of
`formula
`
`animals.
`volume
`
`Tumor
`length
`
`=
`
`volume
`X width2
`
`was
`
`calcu-
`X 0.4.
`
`RESULTS
`
`tially,
`protein
`shown
`
`HEL(cid:1)32
`ng/ml,
`(34).
`
`is
`
`a
`
`34
`
`at
`
`the
`
`a
`
`can
`cell
`carcinoma
`of
`effects
`from MCF-7,
`response
`ng/ml.
`resistant
`
`Ini-
`
`by
`
`in the
`HELcrl
`the
`
`to
`
`of
`
`in
`
`a
`
`1
`
`Table
`to the
`three
`BT-
`are
`N87
`protein,
`MDA-
`HRG-PE
`MDA-
`toxin,
`
`Toxins.
`of HRG-PE
`Isoforms
`of Different
`Activity
`the
`examined
`toxin
`was
`purified
`activity
`of
`each
`the
`As
`using
`[3H]leucine
`labeling.
`assay
`synthesis
`inhibition
`and
`containing
`the HEL(cid:1)31
`toxins
`1,
`in
`Fig.
`chimeric
`0.2-5
`compounds,
`IC50s
`of
`are
`the most
`active
`with
`studies
`which
`are
`similar
`to results
`obtained
`in previous
`(HRG-
`The
`whole
`extracellular
`domain
`fusion
`proteins
`(comparing
`are
`less
`cytotoxic
`and
`less
`specific
`PE38KDEL)
`receptors)
`than
`cells,
`which
`express
`no detectable
`with HUT1O2
`toxins
`made
`with
`the
`HEL
`(HEL-PE38KDEL).
`the
`chimeric
`explanation
`that
`the molecule
`size
`and
`presence
`One
`possible
`of
`other
`structure
`domains,
`such
`as
`the
`immunoglobulin-like
`intact
`domain
`in the
`HRG
`may
`contribute
`the
`reduction
`killing
`and
`specificity.
`The
`cytotoxic
`activity
`of
`the
`(cid:1)3
`of
`fused
`protein
`is more
`than
`100-fold
`less
`than
`that
`in
`isoforms.
`This
`latter
`result
`be due
`to a 10-fold
`reduction
`may
`a]
`binding
`affinity
`for
`the
`isoform
`as
`compared
`to
`the
`(cid:1)3 iso-
`forms
`and
`is
`similar
`to previous
`findings
`(34,
`38). We
`carried
`of
`out most
`the
`following
`experiments
`using
`HEL(cid:1)3ls
`fused
`The
`specificity
`of HEL-PE38KDEL
`PE
`(HEL(cid:1)3l-PE38KDEL).
`and
`was
`verified
`in
`competition
`assay
`the
`cytotoxicity
`blocked
`HEL(cid:1)3l-PE38KDEL
`was
`completely
`low
`concentra-
`concentration
`tion
`and
`partially
`blocked
`at high
`by HRG(cid:1)31
`shown).
`data
`N87
`cells
`(Ref.
`and
`not
`of ErbB-1
`Status
`with
`Cytotoxicity
`and Correlation
`Expression.
`We
`ErbB-4
`(EGFR),
`ErbB-2,
`ErbB-3,
`and
`of HEL(cid:1)3l-PE38KDEL
`in
`the
`next
`evaluated
`cytotoxicity
`and
`breast,
`ovary,
`gastric,
`epi-
`cell
`panel
`of
`cancer
`lines
`from
`soluble
`tetrazolium/formazan
`dermoid
`carcinomas
`using
`the
`cell
`killing
`effects.
`(XTT)
`assay
`to directly
`measure
`summarizes
`data.
`The
`responsiveness
`of
`these
`cells
`IC50
`toxin-mediated
`cell
`killing
`be
`divided
`into
`HRG-PE
`categories.
`Cells
`from
`breast
`cancer
`lines MDA-453,
`474,
`and MDA-36l
`and
`the
`gastric
`cell
`line
`extremely
`sensitive
`to the
`cytotoxic
`the
`fusion
`with
`IC50s
`of <0.01
`ng/ml.
`Cells
`SKBr-3,
`468,
`and
`A43l
`showed
`intermediate
`to
`the
`toxin
`killing,
`with
`IC50s
`of
`0.1-0.5
`MDA-23l,
`and
`SKOV-3
`cells
`are
`to HRG-PE
`4351LCC6,
`of >1000
`ng/ml.
`with
`IC50s
`In general,
`colony-formation
`tumorigenicity,
`with
`in
`vivo
`colonies
`can
`be
`done
`of
`(39)
`recently
`suggested
`al.
`immunotoxin
`efficacy
`is the
`2, MDA-23l
`and
`SKOV3
`and
`MDA-453
`BT-474
`are
`MDA-468
`intermediate.
`able
`to penetrate
`through
`specific
`cell
`killing
`that
`assay.
`
`with
`
`are
`
`et
`
`well
`correlates
`in soft-agarose
`of number
`precise
`quantitation
`and
`system.
`Post
`image
`analysis
`an
`to
`evaluate
`the
`best
`method
`that
`shown
`in Fig.
`clonogenic
`assay.
`As
`cells
`are
`highly
`resistant,
`whereas
`extremely
`sensitive.
`A431
`and
`Therefore,
`the
`chimeric
`toxin
`is
`semisolid
`agarose
`and
`resulted
`in
`the
`is comparable
`to that
`seen
`in the XTT
`
`It
`that
`known
`is
`or more
`members
`one
`recombinant
`toxins,
`
`human
`many
`the EGFR
`of
`including
`TGF-cs
`
`cell
`cancer
`and
`family
`(40-42),
`
`overexpress
`lines
`be
`killed
`can
`anti-ErbB-2
`
`by
`Ab
`
`Downloaded from
`
`on December 31, 2014. © 1998 American Association for Cancerclincancerres.aacrjournals.org
`
`
`Research.
`
`IMMUNOGEN 2158, pg. 3
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`996
`
`Recombinant
`
`Heregulin-Pseudomonas
`
`Exotoxin
`
`100
`
`‘(cid:1)
`
`80
`
`I::e 20
`
`Is
`0.0
`
`I 00
`
`2 80
`
`p\
`
`p(cid:1)
`
`c(cid:1)c(cid:1)c(cid:1)#{231}(cid:1)
`
`c(cid:1).
`
`c(cid:1)
`
`N
`
`N
`
`ng/ml
`
`c(cid:1)
`
`N
`
`N
`
`.
`
`HRGa1
`
`U
`
`HRGfI1
`
`A
`
`HRG(cid:1)32
`
`V
`
`HRGI33
`
`MCF7
`
`A431
`
`HUTIO2
`
`Inhibition
`1
`Fig.
`in MCF7,
`sis
`(adult
`T-cell
`ative)
`cells
`HRG-fused
`HEL-fused
`Data
`points,
`control
`cells
`incorporation.
`
`synthe-
`of protein
`HUT1O2
`A43l,
`and
`leukemia,
`receptor
`neg-
`by
`various
`isoforms
`of
`or
`PE38KDEL
`(top)
`PE38KDEL
`(bottom).
`percentages
`of untreated
`exhibiting
`[3H]leucine
`
`I::2
`
`20
`
`Is
`0.0
`
`#{231}(cid:1)% N
`
`(cid:1) (cid:1)(cid:1)i/
`
`.
`
`HELctI
`
`#{231}(cid:1)% N
`
`ng/mI
`
`U
`
`HEL(cid:1)31
`
`A
`
`HEL(cid:1)32
`
`V
`
`HEL(cid:1)13
`
`of
`
`chimeric
`
`toxin
`
`HEL(cid:1)31-PE38KDEL”
`
`Table
`
`1
`
`Express
`
`ion
`
`of EGFR,
`
`E rbB-2,
`
`ErbB-3,
`
`ErbB-4,
`
`and H RGINDF
`
`and
`
`cytotoxic
`
`a ctivity
`
`Expression
`
`of
`
`Cell
`
`line
`
`EGFR
`
`ErbB-2
`
`ErbB-3
`
`ErbB-4
`
`HRG
`
`Cytotoxic
`
`activity,
`
`IC50 (ng/ml)
`
`-C
`
`+/-
`
`-
`
`-
`
`++++
`
`+
`
`+
`
`+
`
`+++++
`
`++
`
`+++
`
`+
`
`+
`
`+++
`
`+
`
`++
`
`++
`
`+
`
`+++
`
`+
`
`+
`
`+
`
`-
`
`-
`
`-
`
`ND
`
`-
`
`Tumor”
`
`-
`
`+ (E2)
`(E2)
`+
`+++
`(E2)
`+
`
`MDA-453
`BT-474
`MDA-36l
`N87
`MCF-7
`SKBr3
`MDA-468
`A43l
`MDA-23l
`MDA-4351LCC6
`SKOV3
`MCF1OA
`a EGFR,
`
`+/-
`+
`
`++++
`+++++
`
`++
`
`-
`
`-
`
`+
`
`+
`++++
`
`+/-
`
`-
`+
`
`-
`
`-
`
`+++++
`
`+
`++
`
`++
`++
`
`-
`
`++
`
`+/-
`
`++
`
`+/-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`ErbB-2,
`
`and
`
`ErbB-3
`
`expression
`
`was
`
`determined
`
`by Western
`
`blotting
`
`and
`
`0.001
`0.001
`0.001
`0.001
`0. 1
`0.5
`0.5
`0.5
`>1000
`>1000
`>1000
`>1000
`ErbB-4
`
`-
`
`-
`
`ND
`
`+++
`
`ND
`
`-
`
`ND
`immunoblotting
`
`analysis;
`
`+
`
`++++
`++++
`++++
`+++
`
`-
`
`expression
`
`was
`
`determined
`
`by RT-PCR
`
`and Refs.
`
`35
`
`and
`
`by
`36.
`
`+ ,
`
`C._(cid:1) not
`
`relative
`
`immunoblotting,
`
`h Capacity
`
`and
`detectable
`expression
`
`FACS
`
`analysis
`
`(with
`
`NeoMarker’s
`
`Ab-l),
`
`and RT-PCR
`
`analysis;
`
`HRG
`
`expression
`
`was
`
`determined
`
`rate
`
`growth
`tumor
`of
`blot;
`on Western
`level
`on Western
`
`on
`
`+/-
`
`blot;
`
`mice.
`nude
`, detectable
`ND,
`not
`
`only
`
`done;
`
`E2,
`
`extended
`after
`estrogen.
`
`exposure
`
`on Western
`
`blot
`
`or
`
`immunoprecipitation
`
`plus Western
`
`blot;
`
`or HRG
`can bind
`keratinocytes
`
`44),
`(43,
`HRGINDF
`cells
`or
`Siegall
`ErbB-2
`
`a!.
`et
`and
`
`46).
`45,
`(34,
`and activate
`that
`express
`recently
`not
`
`Despite
`ErbB-3
`only
`found
`killed
`by
`
`that
`evidence
`much
`in transfected
`receptor
`receptor
`(47),
`ErbB-3
`the
`expressing
`both
`cells
`that
`the HRGI32-PE4O
`fusion
`
`(45)
`ErbB-3
`
`have
`are
`
`of
`
`protein.
`icity
`ErbB-4
`differences
`same
`cell
`
`(46)
`Jeschke
`However,
`a!.
`correlated
`HRG-PE4O
`their
`The
`expression.
`receptor
`in receptor
`quantitation
`lines
`used.
`is
`also
`possible
`
`reported
`with
`discrepancy
`or
`different
`that
`the
`
`cytotox-
`the
`that
`ErbB-3
`either
`be
`due
`may
`variants
`of
`composition
`
`or
`to
`the
`of
`
`et
`
`It
`
`
`
`Downloaded from on December 31, 2014. © 1998 American Association for Cancerclincancerres.aacrjournals.org
`
`
`Research.
`
`IMMUNOGEN 2158, pg. 4
`Phigenix v. Immunogen
`IPR2014-00676
`
`(cid:1)
`(cid:1)
`

`

`Clinical
`
`Cancer
`
`Research
`
`997
`
`SKOV3
`
`T
`
`T
`
`flh
`
`MDA-468
`
`I
`
`I
`
`T
`
`1000.
`
`800.
`
`600.
`
`400.
`
`200.
`
`0.
`
`2000
`
`-
`
`1600
`
`1200.
`
`800(cid:1)
`
`MDA-231
`
`.j
`
`I 000
`
`800
`
`600
`
`400
`
`200
`
`(cid:1)(cid:1)Hd
`
`0
`
`E
`
`2000
`
`I 600
`
`1200
`
`800
`
`.
`
`.
`
`.
`
`.
`
`A431
`
`(cid:1) (cid:1)
`
`:; (cid:1);
`
`.,
`
`0C
`
`oA (
`
`I)
`0)
`
`0 0C
`
`1(cid:1)(cid:1)
`
`-)
`
`BT474
`
`T
`
`.--
`
`.(cid:1)‘
`
`(cid:1)(cid:1)
`
`#{231}(cid:1).#{231}(cid:1).
`
`400
`
`0
`
`--I
`
`‘.
`‘.
`.,
`
`‘,
`‘,
`‘,
`
`::
`
`_________
`TT(cid:1)
`
`MDA-453
`
`IL
`
`2000
`
`1600
`
`1200
`
`800.
`
`400.
`
`0.
`
`cz(cid:1)(cid:1) #{231}(cid:1).c:y
`
`400.
`
`0.
`
`1000.
`
`800.
`
`600.
`
`400.
`
`200.
`
`0.
`
`0 I
`
`- 0
`
`) Ez
`
`Inhibition
`2
`Fig.
`cells
`of various
`concentrations.
`and colonies
`were
`counted
`alyzer.
`Columns,
`SD.
`
`formation
`colony
`soft-agar
`of
`at various
`by HEL(cid:1)31-PE38KDEL
`for 2 weeks,
`Cells were
`incubated
`that were
`>60
`p.m from three
`dishes
`with Bausch
`and Lomb
`Image An-
`averages
`of
`three
`dishes;
`bars,
`
`with
`of
`of
`
`lines,
`used
`
`to
`leading
`cell
`different
`the
`varies
`receptors
`the ErbB
`of
`series
`a
`IC50s. We
`the
`the
`ratio
`the
`difference
`in
`the
`to screen
`the ErbB
`receptors
`for
`each
`antibodies
`specific
`as
`as well
`by Western
`blotting,
`receptors
`expression
`of ErbB
`Fig.
`(see
`plus Western
`blotting
`combined
`im.munoprecipitation
`EGFR,
`the
`blotting
`analysis
`for
`6).
`Results
`of
`the Western
`of
`these
`phosphorylation
`status
`ErbB-2,
`ErbB-3,
`and
`tyrosine
`in Table
`3 and
`are
`summarized
`receptors
`are
`shown
`in Fig.
`BT-474,
`MDA-
`cells
`(MDA-453,
`of
`The
`highly
`sensitive
`group
`ErbB-3,
`and
`levels
`of ErbB-2,
`high
`361,
`and
`N87)
`expressed
`levels
`the
`N87
`express
`low
`and
`ErbB-4
`receptors.
`BT474
`exposure.
`Cells
`after
`extended
`only
`EGFR,
`which
`are detectable
`toxin
`killing
`to
`the
`HRG-PE
`showing
`intermediate
`response
`and mod-
`high
`levels
`of EGFR
`(MDA-468
`and A431)
`express
`low to mod-
`crate
`levels
`of ErbB-3.
`The MCF-7
`cells
`express
`resistant
`cell
`crate
`levels
`of
`all
`four
`receptors.
`Among
`the
`three
`lines,
`MDA-231
`express
`moderate
`levels
`of
`EGFR
`and
`low
`
`Concentration
`
`of
`
`toxin
`
`(ng/weII)
`
`are
`
`of
`
`ErbB-4
`and
`the ErbB-3
`levels
`but
`the ErbB-2,
`of
`amounts
`MDA-23
`1 cells
`exposure.
`extended
`after
`even
`not
`detectable
`MDA-4531LCC6
`cells,
`HRG/NDF.
`ligand
`the
`also
`express
`only
`moderate
`levels
`of
`to
`killing,
`express
`resistant
`which
`are
`upon
`stimulation
`with
`the
`ErbB-3
`is
`activated
`ErbB-3,
`and
`the
`cells
`(also
`resistant)
`express
`chimeric
`toxin
`(see
`below).
`SKOV3
`or ErbB-4.
`high
`levels
`of ErbB-2
`but
`not ErbB-3
`more
`express
`cell
`lines
`of
`Because
`most
`the
`human
`cancer
`in het-
`and
`differences
`one
`ErbB
`receptor
`family
`member
`than
`affinity
`alter
`the
`binding
`erodimenzation
`of
`these
`receptors
`may
`receptor
`correlate
`the
`and/or
`endocytosis,
`it
`is difficult
`to exactly
`expression
`and
`killing
`efficacy
`of
`the HRG-PE
`toxins.
`We,
`the
`therefore,
`tested
`HEL-PE
`toxin
`killing
`on
`32D cells
`transfected
`with
`EGFR,
`erB-2
`(48),
`erbB-3,
`erbB-4,
`or
`erbB-2
`plus
`erbB-3
`(49).
`None
`the ErbB
`receptors
`were
`detectable
`in the
`parental
`of
`and
`32D
`cells
`The wild-type
`EGFR-,
`erbB-2-,
`or
`erbB-3-
`(49).
`transfected
`32D cells
`are
`resistant
`to the HRG-PE
`killing,
`with
`
`1.
`
`of
`
`Downloaded from
`
`on December 31, 2014. © 1998 American Association for Cancerclincancerres.aacrjournals.org
`
`
`Research.
`
`IMMUNOGEN 2158, pg. 5
`Phigenix v. Immunogen
`IPR2014-00676
`
`(cid:1)
`(cid:1)
`(cid:1)
`

`

`998
`
`Recombinant
`
`Heregulin-Pseudoinonas
`
`Exotoxin
`
`I
`
`(cid:1)s
`
`I
`
`(cid:1) (cid:1)
`$
`
`(cid:1) (cid:1)
`
`/
`
`b(cid:1) (cid:1)
`
`I
`
`(cid:1) (cid:1)-#{176} (cid:1)
`
`WesternBlofting
`EGFRAb
`
`(cid:1)(cid:1)#{246}(cid:1)II(cid:1)e
`
`erbB-2/neuAb
`
`I
`
`..
`
`erbB3Ab
`
`-
`
`-
`
`hsp7OAb
`
`3
`
`Fig.
`ceptor,
`various
`lines
`cinoma),
`ma),
`and
`noma)
`proteins
`these
`8-20%
`expression
`Western
`antibodies
`terials
`against
`protein
`equal
`
`Expression
`ErbB-2,
`human
`and A43l
`N87
`SKOV3
`cells.
`(30
`cells
`
`(cid:1)i.g)
`were
`SDS-PAGE.
`detected
`was
`with
`blotting
`as
`described
`and Methods.”
`the
`endogenous
`was
`used
`as
`loading.
`
`re-
`in
`cell
`car-
`carcino-
`carci-
`cellular
`from
`
`of
`
`EGF
`ErbB-3
`and
`cancer
`breast
`(epidermoid
`(gastric
`(ovarian
`Total
`extracted
`subjected
`Protein
`
`to
`
`by
`
`specific
`in
`“Ma-
`An Ab
`Hsp7O
`a control
`for
`
`levels
`low basal
`HEL(cid:1)31-PE38KDEL
`
`of phosphorylation.
`induced
`tyrosine
`
`Recombinant
`phosphorylation
`
`HRG(cid:1)31
`of
`
`and
`p185
`
`in
`
`(cid:1).LM
`
`manner
`a dose-dependent
`receptor(s)
`erbB
`a strong
`indicating
`(Fig.
`nM-l
`0.1
`SA),
`and HEL(cid:1)3l-PE38KDEL
`HRG
`recombinant
`differences
`in
`phosphorylation
`no
`significant
`HRGI31
`and HEL(cid:1)3l-PE38KDEL
`toxin.
`As
`induction
`of
`ErbB
`receptor(s)
`phosphorylation
`
`various
`
`cell
`
`PE38KDEL
`
`cell
`
`lines
`toxin,
`
`after
`except
`
`treatment
`in MDA-23
`
`over
`agonist
`toxin.
`
`shown
`
`of
`range
`the
`of
`were
`the
`
`SB,
`in
`
`the
`effect
`There
`between
`in Fig.
`found
`was
`nM HEL(cid:1)31-
`10
`with
`1 and MDA-435fLCC6
`
`120
`
`100
`
`80
`
`60
`
`40
`
`0 C0C
`
`)
`
`0 C5
`
`,
`
`U a
`
`,
`a.
`
`20
`
`0
`
`c:(cid:1)(cid:1)’
`
`N
`
`s(cid:1) (cid:1)
`
`‘(cid:1).
`
`N
`
`ng/mi
`
`of HEL(cid:1)3
`Cytotoxicity
`transfected
`stably
`and
`ErbB-4
`receptor.
`
`l-PE38KDEL
`with
`EGFR,
`Data
`points,
`
`32D
`
`on
`ErbB-2,
`averages
`
`wild-type
`ErbB-3,
`of
`
`cells
`ErbB-2
`triplicate
`
`and
`plus
`wells;
`
`4
`cells
`
`Fig.
`32D
`ErbB-3,
`bars,
`
`SD.
`
`lines.
`receptor
`ErbB
`which
`to investigate
`proceeded
`then
`We
`or without
`with
`treated
`protein.
`Cells
`the
`fusion
`by
`activated
`immunoprecipi-
`10 nM for
`10 mm were
`at
`HEL(cid:1)31-PE38KDEL
`ErbB-3,
`specific
`for
`EGFR,
`ErbB-2,
`tated
`with
`antibodies
`then
`blotted
`with
`an antiphosphotyrosine
`and
`ErbB-4
`separately
`results
`are
`shown
`in Fig.
`6. Treatment
`Ab. Representative
`toxin
`induced
`tyrosine
`phosphorylation
`HEL(cid:1)31-PE38KDEL
`and ErbB-4
`receptors
`in MDA-453,
`MDA-361,
`ErbB-2,
`ErbB-3,
`MCF-7,
`and
`SKBr3
`cells.
`Both
`BT-474
`and N87
`lines
`have
`cell
`high
`endogenous
`levels
`of phosphorylated
`ErbB-2,
`ErbB-3,
`and
`ErbB-4.
`Treatment
`with
`HEL(cid:1)3l-PE38KDEL
`resulted
`an
`slight
`increase
`in
`tyrosine
`phosphorylation.
`In MDA-468
`and
`A431
`cells,
`dominant
`phosphorylated
`protein
`migrates
`at
`
`is
`
`and
`
`with
`of
`
`in
`
`the
`
`of
`alone
`
`or
`the
`or
`
`kDa,
`170
`(MDA-23
`have
`no
`visible
`
`region.
`EGFR
`the
`1 , MDA-435/LCC6,
`detectable
`expression
`only
`after
`extended
`
`Among
`
`three
`
`resistant
`
`cell
`
`lines
`
`MDA-23
`SKOV3),
`and
`of ErbB-3
`or ErbB-4
`exposure),
`and
`no
`induction
`
`cells
`
`1
`(ErbB-2
`of
`phos-
`
`is
`
`over
`
`as
`
`shown
`the
`
`4.
`plus
`
`HEL(cid:1)3l-
`The
`in Fig.
`erbB-3-
`erbB-2-
`efficacy
`cytotoxic
`of ErbB-4
`
`at
`IC50s
`PE38KDEL
`erbB-4
`HRG-PE
`ErbB-3
`
`Toxin.
`were
`ability
`which
`
`able
`to
`are
`
`ng/ml
`1000
`on
`active
`is most
`the
`Thus,
`32D cells.
`transfected
`with
`expression
`correlates
`toxins
`of ErbB-2.
`presence
`with
`the
`Phosphotyrosine
`Activation
`Receptor
`purified
`HRG
`recombinant
`The
`and
`to activate
`the HRG
`receptor(s),
`the
`stimulate
`phosphorylation
`of
`known
`express
`ErbB
`
`by Recombinant
`HRG-PE
`toxins
`tested
`their
`breast
`cells,
`and
`have
`
`when
`T47D
`receptors
`
`for
`
`phorylation
`LCC6
`ErbB-3
`resistant
`IC50s
`
`of
`
`by HEL(cid:1)3l-PE38KDEL.
`is observed
`with
`HEL(cid:1)31-PE38KDEL
`treatment
`cells,
`activation.
`However,
`phosphotyrosine
`to
`HEL(cid:1).3l-PE38KDEL-mediated
`1000
`ng/ml.
`SKOV3
`have
`
`high
`
`levels
`
`For MDA-435/
`induced
`is
`line
`with
`no
`
`and
`
`cell
`this
`cytotoxicity,
`of ErbB-2
`
`to
`
`all
`
`four
`
`
`
`clincancerres.aacrjournals.org Downloaded from
`
`on December 31, 2014. © 1998 American Association for Cancer
`Research.
`
`IMMUNOGEN 2158, pg. 6
`Phigenix v. Immunogen
`IPR2014-00676
`
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`

`

`Clinical
`
`Cancer
`
`Research
`
`999
`
`A
`
`B
`
`200
`
`p185
`
`HEL(cid:1)1PE38
`
`HRGf(cid:1)1
`
`I
`
`X
`
`,X,
`
`X
`
`x
`
`g(cid:1)
`
`Tyrosine
`5
`Fig.
`T47D cells
`induced
`centrations
`of HRG(cid:1)31
`and
`PE38KDEL
`(A)
`phosphorylation
`of
`the
`tor(s)
`with
`or without
`HEL(cid:1)3l-PE38KDEL
`various
`cancer
`
`cell
`
`in
`phosphorylation
`con-
`by various
`and HEL(cid:1)3l-
`detection
`erbB
`the
`treatment
`lines
`(B).
`
`of
`
`recep-
`10
`nM
`in
`
`Treatment
`protein.
`or ErbB-4
`ErbB-3
`detectable
`resulted
`in some
`increase
`with HEL(cid:1)3l-PE38KDEL
`but
`not
`in ErbB-4.
`ErbB-3
`phosphorylation
`lines
`cells
`resistant
`whether
`the
`three
`investigate
`To
`tested
`we
`cell
`killing,
`that may
`prevent
`general
`defects
`EGFR;
`for
`(specific
`cytotoxicity
`of TGF-a-PE35KDEL
`erbB-2
`re-
`for
`(specific
`38-40),
`e23(Fv)PE38KDEL
`receptor;
`for
`transferrin
`Ref.
`41), HB21PE4O
`lines
`and
`cell
`in these
`three
`and HEL(cid:1)3l-PE38KDEL
`MDA-435/
`2, MDA-23
`1 and
`As
`shown
`in
`Table
`transferrin
`receptor-mediated
`very
`sensitive
`to the
`SKOV3
`exhibited
`less
`sensitivity
`to
`cells
`and
`e23(Fv)-PE38KDEL,
`despite
`support
`expression.
`These
`data
`the
`of HRG-PE
`toxins
`are mediated
`
`(specific
`
`of
`
`SKOV3
`the
`in ErbB-2
`
`or
`
`the
`1ev-
`that
`the
`
`high
`belief
`through
`
`To determine
`Toxins.
`of Chimeric
`to
`administered
`could
`be
`toxin
`that
`female
`or
`five
`of
`studies,
`groups
`four
`with
`tail
`vein
`injections
`through
`the
`amounts
`of HEL(cid:1)3
`1-PE38KDEL
`diluted
`the
`highest
`dose
`(500
`pUg/kg/mouse),
`At
`3. At
`2501kg/mouse,
`half
`of
`the
`animals
`of
`death
`was
`liver
`failure.
`Liver
`damage
`hemorrhagic
`necrosis
`located
`around
`portal
`
`amount
`the
`animals
`for
`nude
`mice
`100
`(cid:1)il
`of
`in PBS-HSA
`animals
`died
`all
`The major
`died.
`predomi-
`was
`triads.
`Kid-
`
`have
`the
`Refs.
`ceptor;
`47),
`Ref.
`cells.
`N87
`are
`LCC6
`killing.
`cell
`transferrin-PE4O
`els
`of ErbB-2
`specific
`activity
`HRG
`receptors.
`Toxicity
`chimeric
`of
`therapeutic
`were
`given
`increasing
`(0.2%).
`at day
`cause
`nantly
`
`organs
`with
`50
`toxicity
`in
`toxicity
`by
`histo-
`
`as
`
`lung
`no
`
`the
`
`Other
`damage.
`but mild
`similar
`showed
`and
`ney
`treated
`alterations.
`Animals
`pathological
`showed
`sign
`of
`or 25
`p.g/kg/mouse
`showed
`no
`p.g/kg/mouse
`or other
`organ.
`Thus,
`the major
`dose-limiting
`the
`liver
`for HRG-PE
`in mice
`is hepatocellular,
`determined
`logical
`evaluation
`of
`necropsy
`specimens.
`Toxins.
`of
`Chimeric
`Pharmacoki-
`Pharmacokinetics
`were
`deter-
`mice
`in nude
`features
`of
`chimeric
`toxin
`netic
`collected
`samples
`in blood
`by measuring
`the
`toxin
`levels
`mined
`injection
`an
`iv.
`following
`the mice
`at various
`time
`points
`from
`The
`of PBS-HSA.
`200
`(cid:1).tl
`1 (cid:1)i.g
`of HEL