Vol.
`
`1,
`
`/589-1594.
`
`Dee,nber
`
`/995
`
`Clinical
`
`Cancer
`
`Research
`
`1589
`
`Containing
`Immunotoxins
`Endothelial
`Damage
`Human
`Relevance
`to Vascular
`Leak
`
`That
`Exotoxin
`Pseudomonas
`Cells
`in an Antibody-specific
`Syndrome
`
`Le’(cid:1)
`
`Target
`Mode:
`
`Chien-Tsun
`Laboratory
`
`Kuan,
`of Molecular
`
`Lee H. Pai,
`Biology.
`Division
`
`Ira
`and
`of Basic
`
`Pastan’
`Sciences,
`
`National
`
`Cancer
`
`Institute,
`
`NIH,
`
`Bethesda,
`
`Maryland
`
`20892
`
`overt morphological
`undergo
`HUVECs
`B3-LysPE38
`or B3(Fv)PE38.
`ment
`with
`formed
`after
`a 20-h
`exposure
`cells
`are
`These
`studies
`suggest
`that VLS
`in patients
`damage
`caused
`by
`prolonged
`exposure
`tions
`of LMB-1.
`
`treat-
`after
`changes
`the
`between
`Gaps
`2 h.
`not
`after
`but
`is due
`to capillary
`to high
`concentra-
`
`INTRODUCTION
`One
`evolving
`or
`bacterial
`
`plant
`
`approach
`toxins
`to
`
`cancer
`to
`antibodies
`
`treatment
`to make
`
`to
`is
`immunotoxins.
`
`attach
`
`Several
`
`different
`
`and
`
`sonic
`
`of
`
`these
`
`types
`
`are
`
`toxins
`
`that
`
`have
`
`been
`
`of
`
`immunotoxins
`
`have
`
`been
`
`prepared,
`
`now
`
`used
`
`in
`
`clinical
`
`trials
`
`( I , 2).
`
`Some
`
`of
`
`the
`
`to make
`
`immunotoxins
`
`are
`
`ricin
`
`A
`
`protein.
`
`In
`
`chain.
`mutant
`notoxins
`
`blocked
`forms
`have
`
`ricin,
`of PE2
`been
`
`saporin.
`(3-6).
`directed
`
`pokeweed
`Most
`of
`at
`antigens
`
`the
`
`antiviral
`ricin-containing
`present
`on
`the
`
`and
`immu-
`surface
`
`of
`
`bymphomas
`
`and
`
`beukemias
`
`(7,
`
`8).
`
`but
`
`oped
`
`against
`
`solid
`
`tumors
`
`(9-1
`
`1 ).
`
`LysPE38,
`
`a
`
`recombinant
`
`truncated
`
`several
`For
`solid
`form
`of
`PE
`
`have
`been
`tumor
`lacking
`
`deveb-
`therapy,
`the
`cell
`
`binding
`
`domain
`
`( I 2),
`
`has
`
`been
`
`chemically
`
`attached
`
`to murine
`
`monoclonal
`
`antibody
`
`B3,
`
`which
`
`reacts
`
`with
`
`a
`
`carbohydrate
`
`antigen
`
`in
`
`the
`
`Lewis>
`
`family
`
`that
`
`is
`
`present
`
`on
`
`the
`
`surface
`
`of
`
`solid
`
`tumors
`
`( 13).
`
`This
`
`immunotoxin
`
`is
`
`termed
`
`B3-Ly-
`
`many
`
`sPE38
`Phase
`
`causing
`
`utilizing
`chain.
`syndrome
`
`LMB-l
`or
`trial
`I clinical
`been
`VLS
`has
`dose-limiting
`immunotoxins,
`blocked
`ricin,
`is manifested
`
`( 14)
`(15).
`to be
`shown
`toxicity
`including
`more
`decreased
`
`and
`
`is
`
`currently
`
`being
`
`evaluated
`
`in
`
`a
`
`one
`found
`those
`recently,
`serum
`
`of
`
`the major
`in many
`prepared
`(7,
`PE
`albumin
`
`side
`clinical
`with
`8,
`
`effects
`trials
`ricin
`15).
`and
`
`A
`This
`accu-
`
`and,
`by
`
`mulation
`
`edema,
`
`of
`
`fluid
`
`in
`
`the
`
`interstitial
`
`space,
`
`leading
`
`to weight
`
`gain.
`
`and,
`
`in
`
`the
`
`most
`
`severe
`
`cases,
`
`accumulation
`
`of
`
`excess
`
`fluid
`
`in the
`
`lungs
`
`and
`
`pericardiurn.
`
`The
`
`problem
`
`ofeapillary
`
`leak
`
`has
`
`prevented
`
`the
`
`administration
`
`of
`
`the
`
`amount
`
`of
`
`immunotoxin
`
`achieve
`
`maximum
`
`therapeutic
`
`responses.
`
`a
`
`a
`
`in
`
`ABSTRACT
`to
`found
`originally
`was
`(VLS)
`syndrome
`leak
`Vascular
`cancer
`with
`in humans
`toxicity
`dose-limiting
`a major
`be
`A
`ricin
`(ITs)
`containing
`several
`immunotoxins
`treated
`with
`ob-
`been
`VLS
`has
`also
`chain
`or
`blocked
`ricin.
`Recently,
`the murine
`an IT containing
`served
`in patients
`treated
`with
`B3
`coupled
`to LysPE38,
`a re-
`monoclonal
`antibody
`(MAb)
`combinant
`truncated
`form of Pseudomonas
`exotoxin
`(PE)
`A.
`Antibody
`B3
`(IgGik)
`recognizes
`Lewis”
`and
`related
`carbo-
`hydrate
`epitopes
`present
`on many
`human
`solid
`tumors,
`and
`B3-LysPE38
`showed
`excellent
`antitumor
`activity
`in
`nude
`B3
`mice
`bearing
`tumors
`that
`express
`the
`antigen.
`the
`has
`clinical
`trial,
`the
`development
`of VLS
`prevented
`the
`administration
`of
`the
`amount
`of
`IT necessary
`to
`achieve
`blood
`levels
`required
`for
`good
`therapeutic
`responses.
`We
`have
`now investigated
`the
`effects
`of
`several
`PE-based
`on
`ITs
`different
`human
`endothelial
`cell
`lines
`to elucidate
`the mech-
`anism
`of VLS
`induced
`by
`ITs
`containing
`PE. To
`assess
`the
`cytotoxic
`effect
`of
`IT on
`endothelial
`cells,
`various
`ITs were
`incubated
`with
`cells
`for
`incorporation
`of
`h,
`2 or
`20
`and
`the
`[3Hlleucine
`into
`protein
`was measured.
`The
`endothelial
`cells
`were
`studied
`human
`umbilical
`vein
`endothelial
`cells,
`human
`lung-derived
`microvascular
`endothelial
`cells
`(HUVECs),
`hu-
`man
`adult
`dermal
`microvascular
`endothelial
`cells,
`human
`pulmonary
`artery
`endothelial
`and
`human
`aortic
`endo-
`cells,
`thelial
`cells. We
`found
`that
`B3-LysPE38
`(LMB-1),
`both
`chemical
`conjugate
`of MAb
`PE38,
`as well
`as B3(Fv)-
`B3 with
`PE38
`(LMB.7),
`recombinant
`single
`chain
`immunotoxin,
`inhibited
`protein
`synthesis,
`inhibitory
`concentra-
`with
`50%
`600
`1000
`tions
`between
`and
`ng/ml
`for
`20-h
`incubation
`human
`HUVECs,
`lung-derived
`microvascular
`endothelial
`human
`cells,
`and
`adult
`dermal
`microvascular
`endotheliab
`not
`on
`cells
`but
`human
`pulmonary
`artery
`endothelial
`cells.
`The
`cytotoxic
`effect
`was
`specific
`since
`PE38
`itself
`or PE
`coupled
`to
`several
`other
`antibodies
`did
`not
`inhibit
`protein
`synthesis
`in these
`cells
`even
`at
`10,000
`ng/mb.
`Further
`evi-
`dence
`the
`cytotoxicity
`of B3-containing
`ITs
`is due
`to
`that
`B3 binding
`to endothelial
`cells
`comes
`from the
`fact
`specific
`that
`cytotoxicity
`can be blocked
`by excess
`free MAb B3.
`
`the
`
`5/17/95:
`Received
`I To whom requests
`Molecular
`Biology.
`tute,
`NIH,
`37/4E16,
`Phone:
`(301)
`20892.
`
`8/7/95.
`accepted
`7/20/95:
`revised
`addressed,
`should
`be
`reprints
`for
`of Basic
`Sciences,
`National
`Division
`Convent
`Drive,
`MSC
`4255.
`37
`496-4797:
`(301)
`402-1344.
`
`Fax:
`
`at Laboratory
`Cancer
`Bethesda,
`
`of
`Insti-
`MD
`
`lack
`
`ing
`
`necessary
`
`to
`
`VLS
`
`was
`
`not
`
`observed
`
`in
`
`the
`
`preclinical
`
`containing
`
`immunotoxins
`
`in mice
`
`or monkeys.
`
`testing
`
`Because
`
`of
`
`PE-
`
`of
`
`the
`
`of
`
`an
`
`appropriate
`
`mouse
`
`model,
`
`progress
`
`in our
`
`understand-
`
`of
`
`irnmunotoxin-mediated
`
`hypotheses
`
`hypotheses
`Another
`
`been
`have
`is direct
`hypothesis
`
`explored
`of
`damage
`is an indirect
`
`has
`VLS
`to explain
`capillaries
`effect
`
`Several
`delayed.
`been
`of
`these
`the VLS.
`One
`by the
`immunotoxin.
`to the
`production
`
`of
`
`due
`
`2 The
`
`abbreviations
`
`used
`
`are:
`
`PE. Pseudo(cid:1)nonas
`
`exotoxin:
`
`VLS,
`
`vascular
`
`leak
`syndrome:
`cell:
`endothelial
`HAEC,
`human
`microvasculan
`crovascular
`antibody:
`
`5fiC/(cid:1)
`
`A chain:
`RTA.
`human
`HPAEC,
`aortic
`endothelial
`endothelial
`cell:
`endothelial
`cell:
`IC0(cid:1).
`inhibitory
`
`nicin
`
`HUVEC.
`pulmonary
`cell;
`HMVEC-L,
`HMVEC-dAd,
`IL-2,
`interleukin
`concentration.
`
`umbilical
`human
`endothelial
`artery
`human
`lung-derived
`human
`adult
`dermal
`2: MAb,
`monoclonal
`
`vein
`cell:
`
`mi-
`
`Downloaded from
`
`on December 19, 2014. © 1995 American Association for Cancerclincancerres.aacrjournals.org
`
`
`Research.
`
`IMMUNOGEN 2115, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`1590
`
`Immunotoxin-mediated
`
`VLS
`
`other
`or
`has
`VLS
`and
`therapy,
`VLS
`is due
`
`which
`substances
`vasoactive
`observed
`in patients
`been
`that
`it has
`been
`suggested
`to
`damage
`by
`IL-2-aetivated
`
`eapil-
`damage
`high-
`receiving
`in IL-2-treated
`rnonoeytes
`
`Protein
`at 3200
`near
`serum
`After
`
`plated
`or
`until
`human
`jil/well.
`
`Synthesis
`cells/well
`conflueney.
`albumin-PBS
`incubation
`
`Inhibition
`and
`cultured
`and
`or
`Toxins
`added
`were
`at 37#{176}Cfor
`
`the
`
`Assays.
`in 96-well
`controls
`to a final
`indicated
`
`ECs
`for
`in
`
`were
`96 h
`0.2%
`of 200
`(usually
`
`plates
`diluted
`volume
`time
`
`20
`The
`
`each
`h),
`cells
`
`well
`were
`
`was
`subjected
`
`h with
`4
`for
`pulsed
`to a freeze-thaw
`
`[3H]leueine
`cycle
`to lyse
`
`(1
`the
`
`jiCi).
`cells
`
`the
`
`basis
`investigate
`nicin
`containing
`of
`the
`action
`that
`indicated
`immunotoxins,
`of
`interaction
`due
`and
`not
`that
`observed
`in morphology
`
`im-
`al.
`
`receiving
`in patients
`of VLS
`Soler-Rodniguez
`A chain,
`et
`these
`immunotoxins
`on HUVECs.
`the HUVECS
`were
`sensitive
`to the
`the
`and
`that
`cytotoxie
`effect
`was
`ricin
`A chain
`with
`the
`endothelial
`to
`the
`antibody
`to which
`it was
`the HUVECs
`underwent
`rapid
`and
`followed
`by
`inhibition
`of pro-
`
`with
`
`tumors
`
`(B3-
`
`is
`
`to
`
`on
`
`that
`so
`protein.
`vested
`protein
`
`(LKB).
`rated
`age
`MAb
`incubated
`
`could
`we
`After
`glass
`was
`Results
`cpm cells
`of experiments
`B3,
`1 mg/mb
`at 37#{176}Cfor
`
`incorporation
`measure
`thawing,
`freezing
`incorporation
`the
`filters,
`a Betaplate
`by
`quantitated
`as
`the
`calculated
`were
`toxin
`incubated
`without
`performed
`in triplicate.
`MAb
`B3
`was
`added
`30 mm prior
`to toxin
`
`the
`
`and
`and
`
`the
`
`[3H]leueine
`of
`cells
`were
`lysed
`radioactivity
`of
`scintillation
`percentage
`and
`represent
`For
`competition
`each
`to
`addition.
`
`of
`
`into
`har-
`into
`counter
`ineorpo-
`the
`aver-
`with
`and
`time
`
`well
`For
`
`course
`
`study,
`
`the
`
`cells
`
`were
`
`treated
`
`with
`
`different
`
`concentrations
`
`or
`
`24
`
`h. Cells
`
`exposed
`
`to
`
`toxin
`
`to
`
`of
`
`irnmunotoxins
`
`for
`
`2,
`
`4,
`
`8,
`
`12,
`
`24
`than
`less
`for
`further
`incubated
`frozen,
`[3H]leucine,
`Endothelial
`HPAECs
`were
`concentrations
`phology
`of
`
`the
`
`remove
`washed
`h were
`were
`they
`Then
`h.
`24
`until
`counted
`and
`washed,
`thawed,
`Studies
`Cell Morphology
`and
`treated
`grown
`in 35-mm
`dishes
`of B3-LysPE38,
`B3(Fv)-PE38,
`and
`cells
`was
`observed
`after
`2- and
`20-h
`
`the
`
`and
`with
`
`toxin,
`labeled
`(19).
`HUVECs
`and
`various
`with
`RTA. Mor-
`ineubations
`
`eytokines
`The
`laries.
`IL-2
`dose
`patients,
`(16,
`17).
`To
`munotoxins
`(18)
`studied
`Their
`findings
`action
`of
`these
`due
`to a direct
`cell
`membrane
`attached.
`They
`dramatic
`changes
`(18).
`tein
`synthesis
`solid
`patients
`in adult
`trial
`I clinical
`A Phase
`LMB-l
`immunotoxin
`using
`out
`carried
`being
`currently
`The VLS
`appears
`Institute.
`Cancer
`at
`the National
`LysPE38)
`(15).
`To
`investigate
`in this
`trial
`be
`the major
`toxicity
`observed
`several
`different
`have
`exposed
`we
`the mechanism
`of
`this VLS,
`HMVEC-L,
`including
`HUVECs,
`types
`of
`endothelial
`cells,
`and
`other
`HPAECs,
`and HAECs,
`to B3-LysPE38
`HMVEC-dAd,
`The
`results
`indicate
`that
`the
`endothelial
`cell
`immunotoxins.
`damage
`produced
`the
`by
`this
`immunotoxin
`is
`due
`to
`B3
`in
`vitro
`reacts
`of
`the
`immunotoxin
`that
`with
`an antigen
`antibody
`portion
`surface
`of
`endothelial
`cells
`and
`not
`due
`to
`present
`on
`the
`interaction
`of PE38.
`
`an
`
`AND METHODS
`MATERIALS
`HMVEC-L,
`HAECs,
`HUVECs,
`HPAECs,
`Cell Culture.
`purchased
`donors
`were
`isolated
`from
`single
`and HMVEC-dAd
`systems.
`cell
`culture
`Corporation
`EndoPack
`from
`Cbonetics
`growth
`cell
`in endothelial
`at 37#{176}Cin 5% CO2
`They
`were
`grown
`the MCDB
`131
`based
`on
`is a complete
`medium
`medium,
`which
`human
`recombi-
`10 ng/ml
`supplemented
`with
`formulation,
`and
`hydrocortisone,
`2% fetal
`growth
`factor,
`1 jig/ml
`nant
`epidermal
`50
`ng/mb
`amphotericin-B,
`50
`jig/mI
`gentamicin,
`bovine
`serum,
`brain
`extract.
`Endothe-
`bovine
`10 jig/mb
`heparin,
`and
`12 jig/ml
`fifth
`passage
`were
`used
`in these
`hal
`cells
`(ECs)
`in the
`third
`to
`from
`the
`culture
`flasks
`by
`studies.
`ECs
`were
`harvested
`EDTA.
`trypsinization
`in
`the
`presence
`ricin A chain
`The
`Toxins
`and
`Immunotoxins.
`it does
`of
`Inland
`Labs.,
`and
`work
`purified
`by
`J. Fulton
`this
`B3-LysPE38,
`B3(Fv)-PE38,
`contain
`free
`ricin
`B chain.
`not
`MOPC-LysPE38,
`LysPE38,
`e23(dsFv)-PE38,
`anti-Tae(Fv)-
`PE38,
`PEL(cid:1)553,
`and MAb
`B3 were
`prepared
`described
`else-
`where
`(13,
`19-21).
`B3(Fv)-PE38
`is a single-chain
`immunotoxin
`composed
`of
`the
`variable
`(Fv)
`region
`of MAb
`B3 fused
`to PE38
`is
`and
`presently
`undergoing
`clinical
`testing
`(l9).(cid:1)
`e23(dsFv)PE38
`is
`recombinant
`toxin
`composed
`the
`of
`region
`of
`anti-erbB-2
`MAb
`23
`connected
`to PE38,
`in which
`VL
`are
`stabilized
`by
`a
`disulfide
`bond
`(22).(cid:1)
`and
`VH
`is
`a
`recombinant
`immunotoxin
`containing
`Tac(Fv)-PE38
`anti-Tac
`variable
`region
`fused
`to PE38
`(20).
`
`as
`
`Fv
`the
`Anti-
`the
`
`a
`
`antibody
`
`of
`
`used
`
`in
`
`was
`
`using
`
`an Olympus
`
`IMT2
`
`inverted
`
`phase-contrast
`
`microscope.
`
`RESULTS
`To
`on HUVECs.
`B3 MAb
`of
`Specificity
`the
`assess
`cells,
`various
`on
`endothelial
`immunotoxins
`of
`cytotoxie
`effect
`for
`h. As
`20
`with
`HUVECs
`incubated
`were
`immunotoxins
`in
`synthesis
`inhibited
`protein
`B3-LysPE38
`lA,
`shown
`in Fig.
`high
`a very
`ng/rnl.
`Although
`a IC50
`of
`-1000
`HUVECs
`with
`toxic
`this
`is necessary
`to produce
`of B3-LysPE38
`concentration
`excess
`by
`it
`specific
`since
`is blocked
`toxic
`effect
`is
`effect,
`the
`immu-
`an
`(Fig.
`IA).
`Furthermore,
`MOPC-LysPE38,
`MAb
`B3
`known
`no
`made
`with
`the MOPC
`antibody
`that
`notoxin
`on HUVECs
`with
`tumor
`cells,
`had
`cytotoxic
`effect
`reactivity
`of
`the
`IgG 1
`are
`1C). MAb
`B3
`and MAb
`MOPC
`both
`(Fig.
`B3
`antibody
`the
`B3-LysPE38
`is
`composed
`of
`entire
`isotype.
`to LysPE38;
`B3(Fv)-PE38
`is a much
`smaller
`recombi-
`coupled
`immunotoxin
`in which
`the
`binding
`site
`of B3
`in a single-
`nant
`fused
`form
`to
`PE38
`( l9).(cid:1)
`B3(Fv)-PE38
`was
`also
`chain
`is
`ID).
`to HUVECs
`with
`a
`IC(cid:1)0
`-600
`of
`cytotoxie
`ng/ml
`(Fig.
`blocked
`the
`cytotoxieity
`of B3(Fv)-PE38,
`indicating
`B3
`MAb
`also
`binding
`to HUVECs
`via
`the
`antibody
`portion
`it was
`that
`1B).
`immunotoxin
`(Fig.
`In support
`ofthis
`conclusion
`was
`the
`finding
`that
`anti-Tac(Fv)-PE38,
`which
`binds
`to
`the
`subunit
`no
`of
`the
`IL-2
`receptor
`expressed
`on
`human
`monocytes,
`had
`cytotoxie
`effect
`on HUVECs
`(Fig.
`1D and
`Table
`1). Also,
`e23(Fv)PE38,
`recombinant
`irnmunotoxin
`that
`binds
`to
`the
`erbB-2
`receptor,
`did
`not
`protein
`synthesis
`of HUVECs
`inhibit
`even
`the
`extremely
`high
`concentration
`of
`10.000
`ng/ml
`(Table
`1).
`
`has
`
`no
`
`of
`the
`
`p55
`
`a
`
`at
`
`in preparation.
`3 L. Pai, manuscript
`4 Y. Reiter
`and
`I. Pastan,
`unpublished
`
`results.
`
`the
`investigated
`We
`toxins
`on HUVECs
`and
`cytotoxicity
`on HUVECs
`
`cytotoxie
`as
`shown
`with
`
`of
`effect
`in Table
`a ICS() of
`
`other
`several
`2. Native
`-200
`ng/ml.
`
`pro-
`PE did
`This
`
`teins
`show
`
`cytotoxicity
`
`is
`
`due
`
`to
`
`the
`
`presence
`
`of
`
`a2-rnaeroglobulin
`
`recep-
`
`
`
`Downloaded from on December 19, 2014. © 1995 American Association for Cancerclincancerres.aacrjournals.org
`
`
`Research.
`
`IMMUNOGEN 2115, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`Clinical
`
`Cancer
`
`Research
`
`1591
`
`I
`
`of
`
`anti-
`B3
`Specificity
`Fig.
`HUVECs
`HUVECs.
`on
`body
`immunotox-
`with
`incubated
`were
`and
`protein
`inhibi-
`for
`20
`h,
`ins
`was measured.
`of
`the
`cells
`tion
`between
`B3-Ly-
`competition
`) and
`MAb
`excess
`sPE38
`(LMB-l
`competition
`B3
`on HUVECs.
`B.
`between
`B3
`(Fv)-PE38
`(LMB-7)
`and
`excess
`MAE,
`on
`HU-
`VECs.
`comparison
`cytotox-
`of
`C,
`icity
`toward
`HUVECs
`of MOPC-
`LysPE38
`and
`B3-LysPE38.
`comparison
`cytotoxicity
`ward
`HUVECs
`of
`anti-Tac(Fv)-
`PE38
`and
`B3(Fv)-PE38.
`
`A,
`
`D,
`to-
`
`B3
`
`of
`
`Table
`
`I
`
`Cytotoxicity
`
`of
`
`immunotoxins
`
`on
`
`various
`
`human
`
`endothelial
`
`cells
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0I
`
`’-
`4(cid:1)J
`
`C 0U
`
`‘I-
`
`0E0(cid:1)U
`
`of
`
`Time
`incubation
`(h)
`
`20
`2
`20
`2
`20
`2
`20
`2
`
`Proteins
`
`B3-LysPE38
`
`B3(Fv)PE38
`
`ATac(Fv)PE38
`
`e23(dsFv)PE38
`
`(1 ND.
`
`not
`
`done.
`
`A431
`
`2
`
`0.5-1
`2.5
`
`3
`
`HUVEC
`
`1 000
`2,000
`600
`2,300
`> 10,000
`>10,000
`> 10,000
`>10.000
`
`ICS))
`
`(ng/ml)
`
`HAEC
`
`> 10,000
`
`800
`
`HPAEC
`
`> I 0,000
`
`>10,000
`
`> 10.000
`
`> 10.000
`
`HMVEC-L
`
`HMVEC-dAd
`
`---
`
`4,000
`
`750
`
`10,000
`
`7.000
`
`1,200
`
`>10.000
`
`>10,000
`
`ND
`
`ND
`
`> 10,000
`
`tors
`
`(23)
`
`on
`
`the
`
`surface
`
`of HUVECs.
`
`However,
`
`a truncated
`
`form
`
`treated
`
`with
`
`different
`
`concentrations
`
`of
`
`B3-LysPE38
`
`and
`
`of
`
`PE,
`
`LysPE38,
`
`had
`
`10,000
`
`ng/ml
`
`(Table
`
`no
`
`2).
`
`cytotoxic
`
`activity
`
`on HUVECs
`
`even
`
`at
`
`B3(Fv)-PE38
`
`for
`
`various
`
`periods
`
`of
`
`time
`
`(Fig.
`
`2).
`
`and
`
`inhibition
`
`This
`
`presumably
`
`is
`
`because
`
`LysPE38
`
`of
`
`protein
`
`synthesis
`
`was
`
`measured.
`
`In
`
`all
`
`assays.
`
`[3H]Ieucine
`
`was
`
`added
`
`24
`
`h
`
`after
`
`toxin
`
`addition.
`
`When
`
`cells
`
`were
`
`exposed
`
`imrnunotoxin,
`
`they
`
`were
`
`placed
`
`in
`
`immu-
`
`does
`The
`activity.
`
`have
`not
`mutant
`also
`
`a binding
`PEZ(cid:1)553,
`did
`not
`
`domain
`which
`show
`
`any
`
`react
`to
`is defective
`activity
`
`cells.
`endothelial
`with
`in ADP
`ribosylation
`in HUVF.Cs.
`MAb
`83
`
`by
`
`itself
`
`had
`
`no
`
`effect
`
`on HUVECs.
`
`In
`
`confirmation
`
`of
`
`the
`
`findings
`
`Soler-Rodriguez
`of
`to endothelial
`together,
`
`the
`
`ci
`a!.
`with
`in
`
`found
`( I 8), we
`-500
`a ICS() of
`Fig.
`1
`and
`
`cells
`results
`
`that
`ng/ml
`2
`
`was
`RTA
`(Table
`indicate
`
`Table
`
`cytotoxic
`2). Taken
`that
`the
`
`cytotoxie
`
`effects
`
`produced
`
`by
`
`B3-LysPE38
`
`and
`
`B3(Fv)-PE38
`
`for
`
`less
`
`than
`
`24
`
`h
`
`to
`
`notoxin-free
`
`[3Hlleucine
`
`iiwdium
`
`until
`
`24
`
`h
`
`was
`
`reached,
`
`and
`
`then
`
`was
`
`added.
`
`These
`
`results
`
`show
`
`that
`
`activity
`
`caused
`
`by
`
`B3-LysPE3S,
`
`B3(Fv)-PE38,
`
`2-24
`of
`effects
`2
`and
`
`B3-LysPE38
`h. For
`on HUVECs
`increased
`24
`h
`of
`incubation,
`
`and
`
`B3(Fv)-
`approxi-
`whereas
`
`the
`
`the
`
`or
`
`cytotoxic
`
`RTA
`
`in-
`
`are
`
`the
`
`surface
`
`of
`
`endothelial
`
`cells
`
`and
`
`not
`
`due
`
`to
`
`PE38.
`
`Furthermore,
`
`the
`
`cytotoxicity
`
`required
`
`due
`
`to
`
`the
`
`B3
`
`antibody
`
`portion
`
`reacting
`
`with
`
`an
`
`antigen
`
`on
`
`of
`
`an
`
`the
`
`interaction
`
`ADP
`
`ribosy-
`
`creased
`PE38,
`mately
`
`over
`the
`2-fold
`
`period
`the
`cytotoxic
`between
`
`cytotoxic
`
`effects
`
`of RTA
`
`increased
`
`more
`
`than
`
`4-fold
`
`during
`
`this
`
`period.
`time
`B3-LysPE38.
`dependent.
`Capillary
`Are
`to
`
`dAd)
`addition
`
`the
`Thus,
`B3(Fv)-PE38.
`
`results
`
`show
`and RTA
`
`that
`
`cytotoxic
`the
`on HUVEC
`
`effect
`is
`
`of
`time
`
`cells
`
`and HMVEC-
`(HMVEC-L
`Cells
`Endothelial
`Immunotoxins
`Targeting
`Le(cid:1).
`Sensitive
`to
`testing
`effects
`of
`B3-related
`immunotoxins
`
`the
`
`In
`on
`
`lation
`
`of
`activity
`Course
`Time
`Cytotoxicity
`duced
`of
`inhibition
`of
`
`the
`
`protein
`
`toxin.
`B3-LysPE38-
`of
`on HUVECs.
`synthesis
`
`B3(Fv)-PE38-in-
`and
`determine
`the
`kinetics
`
`the
`
`cells
`
`were
`
`To
`in HUVECs,
`
`Downloaded from
`
`on December 19, 2014. © 1995 American Association for Cancerclincancerres.aacrjournals.org
`
`
`Research.
`
`IMMUNOGEN 2115, pg. 3
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`C,)
`C/)
`
`I-z
`C,)z
`-
`
`>-
`
`wI
`
`0a
`
`-
`
`z wz C
`
`-,
`
`w :
`
`i:
`
`1592
`
`Iminunotoxin-mediated
`
`VLS
`
`Table
`
`2 Inhibition
`
`ofprotein
`
`i(cid:1)fltflUilotoXiflS
`
`synthesis
`and
`
`in HUVECs
`toxins
`
`by
`
`various
`
`Proteins
`
`IC50
`
`(ng/mI)
`
`B3-LysPE38
`B3(Fv)-PE38
`NLysPE38
`PE(cid:1)553
`B3 MAb
`PE
`RTA
`a Average
`
`of
`
`three
`
`1,000
`600
`> I 0,000
`>10,000
`>10,000
`210
`500
`
`experiments.
`
`For
`
`each
`
`experiment
`
`performed
`
`in
`
`triplicate,
`
`the HUVECs
`
`were
`
`from
`
`an
`
`individual
`
`single
`
`donor.
`
`human
`cytotoxic
`and
`besides
`a
`
`sources.
`B3(Fv)-
`other
`hu-
`reacts
`
`lym-
`
`HUVECs,
`other
`from
`cells
`endothelial
`studied
`we
`of B3-LysPE38,
`activities
`the
`We measured
`anti-Tae(Fv)-PE38
`on four
`PE38,
`e23(dsFv)-PE38,
`man
`HUVECs.
`Anti-Tae(Fv)-PE38
`endothelial
`cells
`and
`subunit
`present
`on
`leukemias
`with
`the
`IL-2
`receptor
`studied
`were
`HMVEC-L,
`HMVEC-dAd,
`phomas
`(20).
`The
`cells
`and HMVEC-dAd
`are
`and HAECs.
`HMVEC-L
`and HAECs,
`or HPAECs
`and HUVECs
`capillaries
`vessels.
`The
`data were
`from
`as
`large
`blood
`single
`donors
`for HUVECs
`and
`two
`single
`HMVECs,
`HPAECs,
`and HAECs,
`respectively.
`in Table
`1 summarize
`the
`results
`of
`experiments
`type
`of cells were
`incubated
`with
`immunotoxins
`
`prirnar-
`may
`collected
`donors
`
`w -
`
`J
`
`be
`
`for
`
`10000
`
`U-
`
`0 a
`
`-
`C-)
`
`for
`
`HPAECs,
`
`from
`ily
`considered
`from
`four
`HMVEC-L,
`The
`in which
`
`data
`the
`
`h. HUVECs
`20
`2 or
`and
`2000
`1000
`B3(Fv)-PE38
`2300
`ng/ml
`cytotoxicity
`had
`abundant
`ranging
`from
`e23(Fv)-PE38
`IC50s
`were
`
`over
`
`sensitive
`were
`at 20
`and
`ng/ml
`with
`IC50
`of 600
`after
`a 2-h
`exposure.
`of
`these
`agents
`was
`Le”,
`and
`the
`cells
`to
`0.5
`2.5
`ng/ml.
`inhibited
`protein
`10,000
`ng/ml.
`and HMVEC-dAd
`with
`IC50s
`and
`1200
`by
`10,000
`
`IC(cid:1)0s
`with
`to B3-LysPE38
`sensitive
`also
`2 h. They
`were
`exposure
`ng/ml
`after
`a 20-h
`parallel
`experiments,
`In
`examined
`on A43l
`cells
`were
`very
`sensitive
`with
`Neither
`anti-Tae(Fv)-PE38
`synthesis
`in HUVECs;
`
`of
`to
`and
`the
`that
`IC(cid:1)0s
`nor
`their
`
`were
`4000
`of
`ng/ml
`ng/ml
`
`of
`
`sensitive
`also
`7000
`and
`for B3(Fv)-PE38.
`anti-Tae(Fv)-PE38
`
`to B3-
`ng/ml
`for
`The
`or
`
`750
`
`HMVEC-L
`immunotoxins
`based
`B3-LysPE38
`and
`cells
`were
`unaffected
`e23(Fv)-PE38.
`Cells
`detectable
`
`no
`at
`
`with
`resistant,
`more
`were
`HAEC
`from
`derived
`B3-LysPE38
`by
`synthesis
`protein
`of
`inhibition
`B3(Fv)-PE38.
`with
`1000
`mg/ml
`of
`ICst)
`ng/ml
`and
`cells.
`in
`these
`synthesis
`protein
`inhibit
`not
`Anti-Tac(Fv)-PE38
`to B3-LysPE38
`resistant
`completely
`were
`HPAECs
`Interestingly,
`The
`relative
`to anti-Tae(Fv)-PE38.
`as
`as well
`and
`B3(Fv)-PE38
`suggests
`that
`of HPAECs
`and HAECs
`to immunotoxins
`resistance
`less
`Le(cid:1)
`than
`the
`endothelial
`cells
`from the
`large
`vessels
`contain
`vein.
`the
`cells
`endothelial
`from capillary
`beds
`and
`umbilical
`B3(Fv)-
`and
`Morphological
`Changes
`in B3-LysPE38-
`PE38-treated
`HUVECs
`and HPAECs.
`To evaluate
`the
`effect
`HUVECs
`of B3-containing
`immunotoxins
`on
`cell morphology,
`were
`incubated
`with
`various
`concentrations
`of B3-LysPE38
`and
`B3(Fv)-PE38.
`The
`untreated
`HUVEC
`monobayers
`consisted
`of
`tightly
`packed
`cells
`with
`elongated
`shapes.
`Initially,
`HUVECs
`were
`treated
`with
`either
`B3-LysPE38
`or B3(Fv)-PE38
`at 0.01,
`0. 1 , or
`1 jig/mI,
`but
`there
`were
`no morphology
`changes
`ob-
`served
`for
`2-h
`20-h
`incubation
`at
`37#{176}C.Thereafter,
`when
`
`10,000
`
`a
`did
`
`or
`
`10
`
`100
`
`1000
`
`TOXIN,
`
`ng/mI
`
`of
`
`Kinetics
`2
`Fig.
`B3-LysPE38,
`with
`different
`doses
`(#{149}),8 h (LI)’
`ments:
`bars,
`
`inhibition
`B3(Fv)-PE38,
`of B3-LysPE38,
`12 h (U),
`or
`SDs.
`
`of
`
`protein
`and
`RTA.
`B3(Fv)-PE38.
`h (A).
`Points,
`
`treated
`in HUVECs
`synthesis
`with
`were
`treated
`HUVECs
`4 h
`for
`2 h (0),
`or RTA
`mean
`from
`three
`expeni-
`
`24
`
`were
`HUVECs
`no
`at
`10 jig/ml,
`(Table
`3). However,
`B3-LysPE38,
`gaps
`
`treated
`changes
`after
`appeared
`
`either
`with
`cell
`of
`20-h
`
`or B3(Fv)-PE38
`B3-LysPE38
`observed
`after
`shape
`were
`incubation
`with
`B3(Fv)-PE38
`in the
`endothelial
`monolayers.
`
`2 h
`or
`In
`
`only
`
`treatment
`contrast,
`2-h
`after
`to
`exposure
`when
`observed
`tion
`of RTA
`at
`
`with
`incubation,
`RTA.
`Similar
`HUVECs
`1 jig/mI,
`
`RTA
`jig/mI
`10
`gaps
`and
`huge
`morphological
`were
`treated
`which
`was
`
`very
`
`cell
`induced
`found
`were
`changes
`a lower
`with
`similar
`to
`
`rounding
`after
`20-h
`were
`also
`concentra-
`the
`concen-
`
`that
`
`(18)
`
`found
`
`to trigger
`
`rapid
`
`and
`
`Soler-Rodriguez
`tration
`et al.
`changes
`in cell monolayers.
`dramatic
`then
`investigated
`the
`effects
`We
`and RTA
`on the morphology
`PE38,
`summarized
`in
`Table
`3.
`There
`100
`
`of B3-LysPE38,
`of HPAECs.
`The
`no
`effects
`of
`
`B3(Fv)-
`results
`are
`either
`B3-
`
`were
`
`LysPE38
`
`or B3(Fv)-PE38,
`
`at
`
`jig/ml,
`
`on HPAEC
`
`morphology
`
`
`
`clincancerres.aacrjournals.org Downloaded from
`
`on December 19, 2014. © 1995 American Association for Cancer
`Research.
`
`IMMUNOGEN 2115, pg. 4
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`Clinical
`
`Cancer
`
`Research
`
`1593
`
`Table
`
`3
`
`Effect
`
`of
`
`immunotoxins
`
`and toxins
`
`on cell
`
`shape
`
`Time
`
`(h)
`
`B3-LysPE38’4
`
`B3(Fv)-PE38’(cid:1)
`
`RTAa
`
`HUVEC
`
`HPAEC
`
`2
`20
`2
`20
`
`No
`Gaps
`No
`No
`
`effect
`
`effect
`effect
`
`No
`Gaps
`No
`No
`
`effect
`
`effect
`effect
`
`Rounding
`Gaps
`Rounding
`Gaps
`
`cells
`
`cells
`
`(1 HUVECs
`on B3(Fv)-PE38,
`
`and HPAECs
`or
`1 jig/mI
`
`were
`RTA.
`
`treated
`
`with
`
`10 jig/mI
`
`B3-LysPE38
`
`In immunohistoehemical
`the VLS.
`initiates
`vessels
`large
`and
`clinical
`humans
`before
`the
`B3
`on
`MAb
`with
`out
`carried
`studies
`detected
`in the
`capillaries.
`was
`reactivity
`no
`were
`initiated,
`trials
`could
`escape
`detection
`of
`antigen
`amounts
`However,
`small
`yet be sufficient
`to mediate
`binding
`and
`immunocytoehemistry
`and B3(Fv)-PE38.
`cytotoxicity
`of B3-LysPE38
`studies
`with
`B3-LysPE38.
`Prior
`to the
`clinical
`carried
`out
`on Cvnomolgus
`icologicab
`studies
`were
`tolerated
`dose was
`found
`to be 4 mg/kg,
`blood
`level
`of
`approximately
`I 25
`jig/ml.
`
`not
`
`maximum
`an
`initial
`
`by
`and
`
`extensive
`monkeys.
`which
`results
`Even
`at
`
`tox-
`The
`in
`this
`
`at
`
`or
`a 2-h
`after
`rounding
`induced
`of gaps
`formation
`was
`on HPAECs
`together,
`Taken
`by B3(Fv)-PE38
`and
`occurred
`
`ated
`dent
`
`-
`
`only
`
`cell
`1 jig/mb
`RTA
`However,
`exposure.
`20-h
`the
`and
`caused
`after
`a 2-h incubation
`in HPAECs
`1C5() of RTA
`the
`after
`20 h. We
`also
`found
`that
`not
`shown).
`(data
`1000
`ng/ml
`in the 20-h
`assay
`changes
`medi-
`the HUVEC
`cell morphology
`or B3-LysPE38
`were
`concentration
`depen-
`at high
`concentrations
`of
`immunotoxins.
`
`no
`
`likely
`It seems
`observed.
`were
`the VLS
`of
`signs
`level,
`high-dose
`animals,
`these
`of
`is not present
`on the
`capillaries
`that
`the B3 antigen
`and
`esophagus,
`in stomach,
`trachea,
`bladder.
`although
`it
`is present
`humans.5
`in
`are
`which
`also
`the major
`locations
`of
`the
`antigen
`in
`B3-LysPE38
`Toxicological
`studies
`were
`also
`carried
`out
`with
`dose
`mice
`where
`the maximum
`tolerated
`dose
`is 3 mg/kg.
`At
`this
`(2 1).
`there
`is extensive
`damage
`to the
`liver,
`but
`no evidence
`of VLS
`Implications
`for
`Clinical
`Trials.
`In
`a Phase
`I
`study
`ongoing
`in
`patients
`cancers
`expressing
`the
`B3
`
`currently
`
`with
`
`that
`
`that
`and
`
`is
`
`the
`
`as
`
`is
`the
`
`on
`
`of
`
`Lines.
`
`DISCUSSION
`and
`B3-LysPE38
`immunotoxins
`shown
`We
`have
`synthesis
`in cultured
`inhibit
`protein
`specifically
`B3(Fv)-PE38
`umbilical
`vein,
`human
`from
`human
`cells
`derived
`endothelial
`and
`dermal-derived
`mierovaseular
`endotheliurn,
`lung-derived
`exper-
`well
`as endothelial
`cells
`from human
`aorta.
`Competition
`that
`the
`irnents
`and
`comparison
`to other
`immunotoxins
`showed
`cytotoxie
`effects
`of B3-LysPE38
`and B3(Fv)-PE38
`on HUVECs
`are
`specific.
`These
`data
`show
`the L9-related
`B3
`antigen
`present
`on
`endothelial
`cells
`suggest
`this
`basis
`of
`found
`in patients
`treated
`with
`B3-LysPE38.
`B3(Fv)-PE38
`The
`cytotoxic
`effect
`of B3-LysPE38
`and
`a maximum
`HUVECs
`was
`time
`dependent.
`With
`B3-LysPE38
`exposure.
`With
`was
`effect
`observed
`after
`24-h
`continuous
`a
`by
`an
`8-h
`expo-
`B3(Fv)-PE38
`a maximum
`effect
`was
`produced
`of B3(Fv)-PE38
`sure.
`This
`is probably
`rebated
`to the
`instability
`The morphological
`at 37#{176}Cwhere
`it has
`a 1/2
`of
`about
`2 h (24).
`was
`the
`HUVECs
`study
`showed
`that
`no
`dramatic
`change
`of
`observed
`until
`20
`h of
`treatment
`with
`B3-LysPE38
`or B3(Fv)-
`that
`cell
`PE38.
`These
`results
`suggest
`changes
`in
`shape
`are
`protein
`secondary
`to
`inhibition
`of
`synthesis
`caused
`by
`the
`ex-
`posure
`of cells
`to B3-eontaining
`immunotoxins.
`This
`is
`in eon-
`trast
`results
`with
`irnrnunotoxins
`containing
`ricin
`A chain,
`which
`induce
`rounding
`of
`cells
`before
`inhibition
`protein
`(18).
`synthesis
`is
`detected
`We
`Cell
`Different
`with
`Variation
`of Response
`four
`on
`and B3(Fv)-PE38
`analyzed
`the
`activity
`of B3-LysPE38
`was
`line
`HUVECs.
`One
`other
`endothebial
`lines
`besides
`the
`from
`one
`derived
`from
`arteries
`(HPAECs),
`line
`from lung
`derived
`aorta
`(HAECs),
`other
`two
`lines
`were
`and
`(HMVEC-L
`or
`dermal
`endothelium
`with
`was
`observed
`activity
`cytotoxie
`inhi-
`for
`required
`concentration
`but
`the
`on HU-
`required
`that
`was
`greater
`than
`little
`inhibition
`of protein
`synthesis
`was
`(HUVECs,
`HPAECs,
`and HAECs)
`are
`and HM-
`Two
`cell
`lines
`(HMVEC-L
`beds.
`Both
`cell
`lines
`derived
`for
`to both
`types
`of B3 immunotoxins.
`activity
`of B3-LysPE38
`on capillaries
`
`VLS
`
`to
`
`cell
`pulmonary
`and
`the
`rnierovascubar
`Some
`HMVEC-dAd).
`on HAECs,
`B3(Fv)-PE38
`bition
`of protein
`synthesis
`VECs.
`On HPAECs,
`very
`observed.
`Three
`cell
`lines
`derived
`from
`large
`vessels.
`are
`from
`capillary
`
`VEC-dAd)
`
`mierova.seulature
`We
`assume
`
`that
`
`sensitive
`were
`the
`cytotoxie
`
`receiving
`VLS
`found
`antigen,
`jig/kg
`(15).
`from
`doses
`B3-LysPE38
`indicate
`that
`in
`these
`blood
`of
`Measurements
`blood
`levels
`of
`of B3-LysPE38,
`administration
`shortly
`after
`disappears
`from the
`B3-LysPE38
`obtained.6
`are
`500-1000
`ng/rnl
`9 h. Therefore.
`endothelial
`of
`approximately
`blood
`a t(cid:1)
`with
`concentration
`of B3(Fv)-PE38
`to a very
`high
`cells
`are
`exposed
`As
`shown
`in Fig.
`2,
`an
`exposure
`to B3-
`for many
`hours.
`8-h
`LysPE38
`is
`sufficient
`to cause
`significant
`inhibition
`protein
`synthesis
`in HUVECs.
`Thus,
`the
`blood
`levels
`obtained
`in
`pa-
`tients
`correlate
`fairly
`well
`the
`concentrations
`of B3(Fv)-
`with
`PE38
`necessary
`to intoxicate
`HUVECs.
`con-
`high
`to
`capillaries
`of
`the VLS
`is due
`to
`exposure
`available
`are
`strategies
`several
`of B3-LysPE38,
`then
`a
`smaller
`use
`is
`to
`strategy
`this
`side
`effect.
`One
`the
`circu-
`from
`which
`escapes
`weight
`immunotoxin
`in
`contact
`with
`would
`be
`quickly,
`and
`therefore
`a Mr
`62,000.
`and,
`a shorter
`time.
`B3(Fv)-PE38
`has
`from 20
`to 40
`ranges
`monkeys
`and
`mice.
`its
`the
`circulation
`size
`allows
`it
`to
`escape
`from
`better
`penetra-
`probably
`due
`to a combination
`of
`and
`tissues
`and more
`rapid
`excretion.
`There-
`should
`be
`exposed
`to B3(Fv)-PE38
`for
`only
`they
`time
`and may
`not
`damaged
`as
`are with
`be
`If
`capillary
`leak
`syndrome
`observed
`with
`is
`it may
`be
`possible
`to give
`a mixture
`of B3(Fv)-
`polyethylene
`glycol-modified
`MAb
`B3.
`Because
`the
`slowly
`antibody
`escape
`from
`the
`circulation,
`will
`prevent
`the
`of B3(Fv)-PE38
`to
`capillaries.
`binding
`very
`little
`effect
`on binding
`of B3(Fv)-PE38
`to tumor
`are
`outside
`of
`capillary
`bed.
`the
`Immu-
`PE-containing
`Differences
`between
`Ricin-
`and
`notoxins.
`Studies
`shown
`( I 8) have
`by
`Soler-Rodriguez
`et
`a!.
`that
`riein
`A chain
`and
`ricin
`A chain-containing
`imrnunotoxins
`are
`cytotoxie
`to HUVECs.
`These
`effects
`appear
`to be due
`to an
`
`more
`
`If
`centrations
`to
`diminish
`molecular
`lation
`for
`capillaries
`with
`in
`studies
`smaller
`mm.
`Its
`more
`rapidly,
`tion
`into
`tumors
`fore,
`capillaries
`short
`period
`B3-LysPE38.
`B3(Fv)-PE38,
`PE38
`and
`derivatized
`should
`have
`that
`
`of
`
`a
`
`it
`but
`cells
`
`we
`
`have
`at
`
`the
`that
`ranging
`levels
`
`occurs
`10
`
`patients
`in
`100
`to
`patients
`
`of
`
`I. Pastan,
`and
`L. Pai,
`S M. Willingham.
`6 L. Pai
`and
`I. Pastan,
`unpublished
`data.
`
`unpublished
`
`data.
`
`Downloaded from
`
`clincancerres.aacrjournals.org
`
`on December 19, 2014. © 1995 American Association for Cancer
`Research.
`
`IMMUNOGEN 2115, pg. 5
`Phigenix v. Immunogen
`IPR2014-00676
`
`(cid:1)
`

`

`1594
`
`Immunotoxin-mediated
`
`VLS
`
`of
`
`shown
`As
`cells.
`the
`with
`A chain
`ricin
`interaction
`sensitive
`are
`that HUVECs
`findings
`their
`we
`confirmed
`Furthermore,
`at 24 h of 500
`ng/mI.
`a IC50
`A chain
`with
`which
`the morphology
`of HUVECs
`on
`a
`rapid
`effect
`for
`cells
`incubation
`of
`that
`toxin
`with
`the
`observed
`after
`independent
`less.
`It
`has
`been
`suggested
`that
`this
`effect
`is
`of HUVECs
`protein
`synthesis
`(16).
`In contrast,
`a 20-h
`exposure
`to
`observe
`to
`B3-LysPE38
`or
`B3(Fv)-PE38
`was
`required
`a time
`at
`changes
`in morphology,
`and
`these
`changes
`occurred
`when
`protein
`synthesis
`was markedly
`inhibited.
`The mechanism
`of
`alteration
`of HUVECs
`by
`ricin-containing
`immunotoxins
`PE-containing
`immunotoxins
`appears
`to be
`different.
`We
`are
`currently
`conducting
`preclinical
`studies
`
`2,
`in Fig.
`to riein
`RTA
`has
`can
`be
`2 h or
`of
`
`and
`
`with
`
`recom-
`
`binant
`
`irnmunotoxins
`
`containing
`
`(20),
`
`region,
`erbB-2,
`these
`not
`of
`
`anti-Tac(Fv)-PE38
`e23(Fv)-PE38KDEL
`agents
`should
`begin
`the VLS
`observed
`with
`the
`antibody
`or
`toxin
`
`the
`
`variable
`antibody
`anti-Tac
`the
`directed
`an
`immunotoxin
`and
`studies
`Phase
`I clinical
`(25).
`whether
`a year
`and
`clarify
`within
`B3-LysPE38
`is due
`to an interaction
`with
`endothelial
`cells
`in patients.
`
`at
`with
`or
`
`ACKNOWLEDGMENTS
`We
`thank
`E. Lovelace
`and
`R. Kneitman
`for
`Bailon
`for e23(dsFv)PE38.
`We
`also
`assistance.
`
`endothelial
`the
`for help with
`providing
`anti-Tac(Fv)-PE38,
`thank A. Jackson
`and J. Evans
`
`culture,
`cell
`and Y. Reiter
`for editorial
`
`P.
`
`therapy
`Immunotoxin
`and
`S. Rosenberg
`1 1 1-135.
`Philadelphia:
`
`of ma-
`(eds.),
`J. B.
`
`but
`
`J. Chem. Oncol..
`
`lym-
`killer
`in
`albumin
`for
`vascular
`
`N.,
`immu-
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`
`del Rio, M.,
`L.,
`Spitler
`10.
`Miller
`L. L., Harkonen,
`Misehak,
`R.
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`L. G., Hudson,
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`
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`
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