Humanized M195 monoclonal antibody conjugated to
`recombinant gelonin: an anti-CD33 immunotoxin with
`antileukemic activity.
` 
`L C Pagliaro, B Liu, R Munker, et al.
`Clin Cancer Res  
`
`1998;4:1971-1976.
`
`Updated version
` 
`
`Access the most recent version of this article at:
`http://clincancerres.aacrjournals.org/content/4/8/1971
` 
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`Downloaded from Downloaded from
`
`on October 7, 2014. © 1998 American Association for Cancerclincancerres.aacrjournals.org on October 7, 2014. © 1998 American Association for Cancerclincancerres.aacrjournals.org
`
`
`
`
`
`Research. Research.
`
`IMMUNOGEN 2160, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`Vol.
`
`4,
`
`1971-1976,
`
`August
`
`1998
`
`Clinical
`
`Cancer
`
`Research
`
`1971
`
`Humanized
`Gelonin:
`
`Antibody
`Monoclonal
`M195
`An Anti-CD33
`Immunotoxin
`
`Conjugated
`with
`Antileukemic
`
`to Recombinant
`Activity’
`
`Lance
`
`C.
`
`Pagliaro,2
`
`Baoshun
`
`Liu,
`
`Michael
`
`AndreefT,
`
`Reinhold
`
`Emil
`
`Michael
`
`Munker,
`J Freireich,
`G.
`Rosenbium
`
`David
`
`A.
`
`Scheinberg,
`
`and
`
`J. F., M. G. R.]
`E.
`[L. C. P.. R. M.. M. A.,
`of Medicine
`Divisions
`of Texas
`M. D.
`[B. L.].
`The
`University
`Medicine
`Laboratory
`77030,
`and
`the Department
`Center,
`Houston,
`Texas
`Cancer
`Anderson
`Sloan Kettering
`Cancer
`Leukemia
`Service. Memorial
`of Medicine.
`Center.
`New York. New York
`10021
`[D. A. 5.1
`
`and
`
`We
`immunotoxin.
`gated
`to recombinant
`should
`be
`considered
`
`conclude
`gelonin
`for
`clinical
`
`that
`has
`
`humanized
`antileukemic
`testing
`in Phase
`
`M195
`activity
`I trials.
`
`conju-
`and
`
`INTRODUCTION
`The
`development
`for
`the
`
`potential
`
`of
`
`cell-targeted
`of
`hematopoietie
`
`treatment
`
`cytotoxie
`
`suggested
`
`by
`
`clinical
`
`responses
`
`variety
`
`geting
`
`of
`
`immunoconjugates
`
`cell
`
`surface
`
`antigens
`
`and
`
`in Phase
`observed
`( 1-3). Monoelonal
`growth
`factors
`
`agents
`malignancies
`I trials
`antibodies
`
`holds
`as
`a
`
`with
`tar-
`
`such
`
`as
`
`interleu-
`
`kin
`
`2
`
`fused
`
`or
`
`coupled
`
`to
`
`toxins
`
`have
`
`been
`
`studied
`
`as
`
`potential
`
`therapeutic
`
`agents,
`
`but
`
`success
`
`has
`
`been
`
`limited
`
`by
`
`obstacles
`
`as
`
`such
`adverse
`
`immunogenicity,
`effects
`due
`
`to the
`
`of
`
`lack
`extreme
`
`specific
`toxicity
`
`tumor
`of plant
`
`antigens,
`and
`
`and
`bacterial
`
`design
`address
`cells
`
`new
`of
`these
`in
`
`immunotoxins
`CD33
`problems.
`normal
`hematopoiesis
`
`requires
`is found
`and
`
`the
`on
`has
`
`ABSTRACT
`HuM195-
`immunotoxin
`characterized
`recently
`The
`an-
`monoclonal
`anti-CD33
`of a humanized
`consists
`gelonin
`gelonin.
`toxin
`to
`the
`single-chain
`plant
`conjugated
`tibody
`that
`ex-
`cells
`immunotoxin
`to hematopoietic
`of
`the
`Binding
`demon-
`been
`has
`differentiation
`antigen
`CD33
`the
`press
`macti-
`ribosomal
`to
`in cytotoxicity
`due
`results
`and
`strated
`acute
`with
`patients
`Blast
`cells
`from
`most
`by
`gelonin.
`vation
`stem
`normal
`whereas
`myelogenous
`leukemia
`express
`CD33,
`do not. We
`cells
`necessary
`for maintenance
`of hematopoiesis
`recombinant
`ge-
`asked
`whether
`an
`immunoconjugate
`using
`lonin
`rather
`than
`plant
`gelonin
`to acute
`myelogenous
`is toxic
`ob-
`and
`leukemia
`(AML)
`cell
`lines
`primary
`AML
`blasts
`the
`exposed
`tamed
`from
`patients
`and
`to
`immunotoxin
`in
`cell
`The
`lines
`HL6O,
`OCIJAML2,
`and OCIJ
`vitro.
`CD33”0”
`decreased
`AMLS
`showed
`proliferation
`when
`exposed
`to im-
`24-72
`munotoxin
`for
`h. The
`CD33(cid:1)
`cell
`line OCIJAML3
`resistant
`was
`relatively
`to HuM195,
`and
`all
`cell
`lines
`were
`resistant
`to equimolar
`concentrations
`of unconjugated
`anti-
`body
`gelonin.
`Primary
`blast
`cultures
`from
`pa-
`and
`seven
`tients
`with AML
`had CD33
`detectable
`on 75.7-99.8%
`of cells
`by flow cytometry,
`and
`all
`showed
`dose-dependent
`decreases
`the
`in clonogenic
`cell
`survival
`during
`24-h
`incubation
`with
`immunotoxin.
`Cells
`selected
`for
`low CD33
`expression
`by cell
`sorting
`or by prolonged
`incubation
`with
`immunotoxin
`could
`reexpress
`CD33
`at baseline
`levels
`and
`remained
`sensitive
`to
`
`this
`
`revised
`7/16/97:
`of publication
`of page
`charges.
`accordance
`
`5/13/98.
`accepted
`5/5/98:
`were
`defrayed
`article
`of
`this
`This
`article
`must
`therefore
`with
`I S U.S.C.
`Section
`
`be
`
`part
`in
`hereby
`I 734
`
`the
`by
`marked
`solely
`
`to
`
`for Research
`CA16672,
`Society
`at
`the
`Phila-
`of
`the
`I 997
`
`Society
`at
`and
`Research.
`
`in
`fact.
`Foundation
`the Clayton
`by
`in part.
`conducted.
`CA55164,
`Grants
`CA55349,
`by NIH
`in part.
`is
`a
`recipient
`of
`an American
`Cancer
`L. C. P.
`Development
`Award.
`Presented.
`in part.
`Career
`the American
`of Clinical
`Oncology.
`1047)
`(abstract
`the
`Annual
`Meeting
`for
`Cancer
`San
`Diego,
`CA,
`
`Received
`The
`costs
`payment
`advertisement
`indicate
`I Research
`and
`supported,
`and
`CA57639.
`Clinical
`Oncology
`of
`Annual
`Meeting
`1996
`delphia.
`PA.
`American
`Association
`(abstract
`569).
`2 To whom
`Medicine,
`Center.
`
`for
`requests
`Box
`13, The
`1515
`Holcombe
`
`at Division
`addressed,
`be
`should
`reprints
`M. D. Anderson
`Cancer
`of Texas
`University
`Blvd.,
`Houston,
`TX 77030.
`
`of
`
`abbreviations
`3 The
`recombinant
`gelonin:
`tibody
`conjugated
`CFU.
`colony-forming
`
`are: AML.
`used
`HuMI95-rGel,
`rOd:
`FACS.
`unit.
`
`to
`
`acute myelogenous
`humanized
`M195
`fluorescence-activated
`
`leukemia:
`monoclonal
`cell
`
`rOd,
`an-
`sorting:
`
`
`
`Downloaded from on October 7, 2014. © 1998 American Association for Cancerclincancerres.aacrjournals.org
`
`
`Research.
`
`toxins
`use
`the
`
`The
`(4).
`used
`that
`strategies
`of
`surface
`of myeloid
`
`structural
`the
`ber
`of
`of CD33
`
`and
`binding
`sialoadhesin
`is
`not
`known
`
`that
`
`characteristics
`family
`of proteins
`but may
`involve
`
`identify
`The
`(5).
`adhesion
`
`as
`
`it
`exact
`or
`
`a mem-
`function
`cell-cell
`
`communication.
`
`CD33
`
`is
`
`also
`
`expressed
`
`on
`
`blast
`
`cells
`
`in most
`
`eases
`
`of
`
`AML3
`
`but
`
`is
`
`not
`
`expressed
`
`on
`
`the
`
`earliest
`
`normal
`
`(6).
`the
`may
`that
`agents
`(murine)
`
`cells
`at
`
`surface
`be
`useful
`8). The
`HuM195
`
`(7,
`and
`
`stem
`hematopoietie
`is a
`target
`CD33
`AML
`with
`therapeutic
`M195
`
`patients
`targeted
`antibodies
`
`clinical
`localizes
`
`AML
`with
`HuMl95
`
`cells
`
`in
`of
`
`in
`
`leukemie
`development
`monoclonal
`in
`are
`now
`M 195
`patients
`whereas
`
`of most
`the
`for
`anti-CD33
`(humanized)
`‘ 1-Radiolabeled
`the
`for
`trials
`to peripheral
`marrow
`blast
`cells
`blood
`and
`monoclonal
`is a murine
`(7). Ml95
`lgG2a,
`consists
`of murine
`complementarity-determining
`
`treatment
`
`of AML.
`
`‘
`
`re-
`
`and
`
`for
`of
`to
`
`of
`
`gions
`constant
`affinity
`binding
`preserved
`HuMI95
`(9).
`regions
`(cid:1)3 half-life
`a serum
`and
`has
`immunogenic
`is not
`but
`CD33
`not
`appear
`with HuMl95
`did
`ofgelonin
`h (8). Conjugation
`42
`( 10)
`recently
`interfere
`with
`McGraw
`to CD33.
`binding
`a!.
`M 195-gelonin,
`is a conjugate
`characterized
`humanized
`toxin
`gelonin,
`which
`is
`reactive
`HuMl95
`the
`plant
`and
`against
`CD33P(cid:1)s
`myeloid
`leukemia
`line
`HL6O
`in
`vitro
`
`genetically
`
`grafted
`
`to
`
`a
`
`human
`
`IgG
`
`1
`
`framework
`
`has
`
`the
`
`(10).
`
`et
`which
`and
`cell
`
`was
`
`toxin
`Gelonin
`rnultifloruin.
`Geloniurn
`that
`inactivates
`the
`adenine
`N-glycoside
`Unlike
`dual-chain
`hydrate-binding
`
`It
`ribosomal
`bonds
`toxins
`domain
`
`isolated
`originally
`30,000
`is
`a Mr
`subunit
`60S
`sequence-specific
`as
`ricin,
`gelonin
`itself,
`cannot
`
`of
`
`seeds
`the
`from
`protein
`single-chain
`rRNA
`by
`cleaving
`( 1 1).
`fashion
`a earbo-
`lacks
`bind
`to cells
`and
`
`a
`
`by
`
`in
`such
`and,
`
`IMMUNOGEN 2160, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`(cid:1)
`

`

`1972 HuMI95-rGel
`
`Antileukemic
`
`Activity
`
`antibody,
`synthesis
`cloning,
`and
`protein
`(rGel)
`(12).
`toxin
`The
`in contrast
`to
`
`internalizing
`to an
`Conjugated
`nontoxic.
`relatively
`is
`inhibiting
`protein
`is highly
`toxic,
`gelonin
`however,
`the
`sequencing,
`we
`reported
`Recently,
`irreversibly.
`of
`recombinant
`gelonin
`a
`expression
`bacterial
`properties
`to the
`natural
`with
`identical
`biological
`used
`the
`recombinant
`toxin
`studies
`reported
`herein
`used
`natural
`gebonin.
`previous
`studies
`that
`activity
`have
`to
`was
`found
`previously
`HuM195-gebonin
`( 10). We
`asked
`against
`cell
`line
`HL6O
`the myeboid
`vitro
`in
`with
`HuMl95-
`whether
`could
`be
`demonstrated
`similar
`activity
`patients
`with
`AML
`obtained
`freshly
`from
`blast
`cells
`using
`rGel
`A clonogenic
`culture
`and maintained
`in liquid
`culture
`medium.
`on AML
`blast
`progen-
`system
`was
`chosen
`to measure
`the
`effect
`HuM l95-rGel
`is
`active
`itor
`specifically.
`We
`report
`that
`against
`AML
`cell
`and
`primary
`cultures
`and
`that
`this
`effect
`is
`specific
`to CD33(cid:1)(cid:1)(cid:1)S
`cells.
`
`cells
`
`lines
`
`20
`
`AND METHODS
`MATERIALS
`prepared
`was
`HuM195
`of
`Immunotoxin.
`Preparation
`Design
`supplied
`by Protein
`and was
`generously
`as described
`(9)
`in Esch-
`rGel was
`expressed
`Labs,
`Inc.
`(Mountain
`View,
`CA).
`Conjuga-
`previously
`(12).
`and
`purified
`as described
`erichia
`coli
`described
`performed
`as
`also
`of HuMl95
`with
`rGeb
`was
`tion
`3-(2-pyridylthio)propi-
`previously
`(10).
`Briefly,
`N-succinimidyl
`buffer,
`phosphate
`onate-modified
`HuMl95
`in
`100 mi(cid:1)i
`sodium
`0.5
`of
`excess
`a 5-molar
`with
`(pH 7.0), was mixed
`EDTA
`mM
`to 7.0 with
`adjusted
`pH was
`2-iminothiolane
`modified
`rGel.
`The
`0.5 M triethanolamine
`the mixture
`8.0),
`and
`(pH
`hydrochloride
`To
`stop
`the
`nitrogen.
`for
`was
`incubated
`h
`at
`4#{176}Cunder
`reaction,
`iodoacetamide
`was
`added
`to a final
`concentration
`of 2
`and
`incubated
`1 h at
`room
`temperature.
`The
`reaction
`for
`mM
`mixture
`was
`purified
`on a Sephacryl
`S-300
`gel
`filtration
`column
`equilibrated
`with
`20 mrvi Tris
`and
`50 m(cid:1)vi NaC1
`(pH
`7.4).
`The
`fractions
`containing
`immunotoxin
`and
`unreacted
`antibody
`were
`pooled
`and
`then
`loaded
`on
`a Cibacron-blue-Sepharose
`CL-6B
`column
`equilibrated
`with
`Tris
`buffer
`to
`remove
`unconju-
`gated
`antibody.
`Purified
`immunotoxin
`was
`eluted
`20 mrvi
`dialyzed
`Tris/2
`M NaCb/pH
`7.4
`buffer
`and
`against
`OCIJ
`Cell
`Lines.
`HL6O,
`OCI/AM2,
`OCIJAML3,
`supple-
`were maintained
`in a minimal
`essential
`medium
`AMLS
`Inc.,
`with
`10% fetal
`bovine
`serum (Life
`Technologies,
`mented
`Island,
`NY),
`at
`37#{176}Cwith
`Grand
`CO2
`in
`a humidified
`5%
`Maintenance
`cultures
`of OCI/AML5
`were
`also
`sup-
`incubator.
`with
`0.1 ng/ml
`plemented
`recombinant
`granubocyte/macrophage
`MN).
`colony-stimulating
`factor
`(R and D Systems,
`Minneapolis,
`in-
`Collection
`Patient
`Samples.
`After
`obtaining
`of
`formed
`consent,
`approximately
`10 ml of peripheral
`blood were
`collected
`in sodium
`heparin
`patients
`diagnosed
`with AML
`from
`and
`hospitalized
`the
`of Texas
`M. D. Anderson
`at
`University
`de-
`Cancer
`Center.
`Samples
`immediately
`processed
`as
`were
`scribed
`previously
`(13).
`samples
`diluted
`2: 1 with
`Briefly,
`were
`(without
`calcium
`or magnesium),
`then mononuclear
`cells
`HBSS
`were
`isolated
`by density
`gradient
`centrifugation.
`T lymphocytes
`were
`separated
`from
`the
`mononuclear
`cells
`by
`sheep
`RBC
`rosetting,
`followed
`by
`density
`gradient
`centrifugation.
`This
`pro-
`cedure
`yields
`a cell
`population
`that
`is >90%
`blasts.
`For main-
`tenance
`culture,
`cells
`were
`kept
`in a minimal
`essential
`medium
`with
`10% fetal
`bovine
`serum,
`1 ng/ml
`granulocyte/macrophage
`
`the
`with
`PBS.
`and
`
`and
`
`colony-stimu-
`granulocyte
`0. 1 ng/ml
`factor,
`colony-stimulating
`factor
`(R and D Systems).
`stem cell
`1 ng/mb
`bating
`factor,
`AML
`Cultures.
`Clonogenic
`cell
`lines
`or patient
`samples
`for
`24-72
`h with
`HuMl95-
`culture
`in
`liquid
`incubated
`were
`the
`concentrations
`indicated,
`at
`rGel
`and
`or HuM195
`rGel,
`rGel,
`the
`per ml
`at
`the
`beginning
`of
`density
`of one million
`a cell
`with
`twice
`in a minimal
`essen-
`experiment.
`Cells
`were
`then
`washed
`solution
`(15%
`fetal
`bovine
`tial medium,
`resuspended
`in plating
`factors,
`0.8% methylcellulose),
`and
`plated
`
`serum,
`
`growth
`
`and
`
`in
`
`at
`
`a
`
`for
`cells,
`
`(Linbro,
`plates
`microtiter
`96-well
`in
`ml/well,
`0.1
`quadruplicate,
`to plating
`added
`suspension
`of cell
`amount
`PA). The
`Horsham,
`liquid
`culture
`from
`to equal
`volumes
`of
`corresponded
`solution
`taken.
`Colonies
`consisting
`of
`>20
`cells
`cells
`had
`been
`which
`counted
`after
`S to 7 days
`of
`incubation
`37#{176}Cwith
`5%
`were
`To
`CO2.
`determine
`the
`effect
`of
`cryopreservation,
`cells
`were
`to HuMl95-rGel
`(1 nM)
`for
`exposed
`resuspended
`h, washed,
`24
`and
`tubes,
`with
`10% DMSO
`and
`50% FBS
`in 6.0-mb
`in medium
`were
`-70#{176}Cfreezer
`for
`a minimum
`of
`24
`h. Cells
`placed
`in a
`bath,
`thawed
`by
`immersion
`in
`37#{176}Cwater
`then
`quickly
`washed,
`incubated
`at 37#{176}Cfor
`24 h, washed
`again,
`and
`plated
`in
`methylcellubose.
`Control
`cells
`were
`incubated
`24
`h at
`the
`as
`same
`temperature
`and
`density
`thawed
`then
`plated.
`cell
`and
`Flow
`Cytometric
`Analysis
`Sorting.
`Bone
`marrow
`AML
`aspirates
`from
`patients
`with
`were
`analyzed
`CD33
`for
`protocol
`expression
`using
`a standardized
`in the Flow
`Cytometry
`of Texas
`laboratory
`at
`the University
`M. D. Anderson
`Cancer
`of
`Medicine.
`Center,
`Division
`of Laboratory
`CD33
`expression
`with
`a CD33
`lines
`cell
`was
`detected
`using
`antibody
`labeled
`detect
`San
`CA).
`phycoerythrin
`(Becton
`Dickinson,
`Jose,
`To
`F(ab)2
`goat
`bound
`immunotoxin,
`FITC-labeled
`anti-human-IgG
`Francisco,
`from
`antibody
`fragments
`were
`obtained
`Caltag
`(San
`CA)
`done
`using
`and
`used
`at a dilution
`of
`1 :5. The
`analysis
`was
`on
`a FACScan
`(Becton
`Dickinson,
`San
`Jose,
`CA),
`gated
`live
`cells
`by
`criteria.
`For
`all
`staining
`procedures,
`nonspecific
`scatter
`binding
`controlled
`for
`using
`an
`irrelevant
`antibody
`of
`was
`same
`and was
`subtracted.
`Incubations
`were
`performed
`isotype
`ice
`on
`0.2%
`BSA,
`and
`NaN3).
`FACS
`sorting
`was
`(PBS,
`performed
`under
`sterile
`conditions
`using
`a FACS
`Vantage
`(Bee-
`ton Dickinson,
`San
`Jose,
`CA).
`
`the
`
`0.1%
`
`RESULTS
`Lines
`for Cell
`of HuM195-rGel
`Cytotoxicity
`CD33.
`HL6O colony
`Express
`That
`was
`inhibited
`(Fig.
`1), with
`in
`a dose-dependent
`by HuMl95-rGel
`72-h
`incubation
`obtained
`in clonogenic
`cells
`log
`reduction
`with
`results
`pub-
`is
`consistent
`with
`5 nM immunotoxin.
`This
`Colonies
`did
`lished
`previously
`with
`HuM195-gebonin
`(10).
`that
`form after
`exposure
`to HuM 195-rGeb
`smaller
`than
`colonies
`were
`from untreated
`cells
`or cells
`exposed
`to unconjugated
`antibody
`and
`rGel
`or
`free
`rGel
`alone,
`indicating
`a degree
`growth-
`on
`inhibitory
`effect
`all
`cells
`in the
`culture.
`The AML
`cell
`lines
`OCIJAML2
`and OCIJAML5
`are CD3Y(cid:1)(cid:1)S
`and
`showed
`decreased
`colony
`formation
`in response
`to 48-h
`incubation
`with
`HuM 195-
`but were
`growth
`inhibited
`by equivalent
`concentrations
`rGel
`unconjugated
`HuMl95
`and
`rGel.
`OCIIAML3
`does
`not
`of
`press
`CD33,
`colony
`formation
`was
`inhibited
`HuM195-rGel
`not
`shown).
`
`Is Specific
`formation
`manner
`after
`
`a
`
`of
`
`not
`
`cx-
`by
`
`not
`
`and
`(data
`
`Downloaded from
`
`on October 7, 2014. © 1998 American Association for Cancerclincancerres.aacrjournals.org
`
`
`Research.
`
`IMMUNOGEN 2160, pg. 3
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`Clinical
`
`Cancer
`
`Research
`
`1973
`
`I
`Table
`antibody
`
`immunotoxin
`IC50 of
`and
`toxin
`for HL6O
`
`versus
`and
`seven
`
`unconjugated
`AML
`patient
`
`monoclonal
`samples
`
`IC50 (nM)”
`
`24-h
`
`incubation”
`
`72-h
`
`incubation”
`
`Immunotoxin
`
`HL6O
`1
`Patient
`2
`Patient
`Patient 3
`Patient
`4
`Patient
`5
`Patient
`6
`Patient
`7
`a Fifty
`
`0.3
`1.0
`4.0
`6.0
`30
`1.0
`10
`9
`% inhibitory
`
`Immunotoxin
`
`0.02
`5.0
`0.1
`
`HuMl95
`rOd
`+
`
`100
`20
`3.0
`
`HuMb95
`rOd
`+
`
`>10
`100
`100
`
`200
`
`concentration
`
`estimated
`
`from plot
`
`of colonies
`
`of
`
`immunotoxin
`
`or
`
`unconjugated
`
`equimo-
`
`100
`
`le
`
`0 C8 0
`
`=
`
`a .(cid:1) 08
`
`o:(cid:1)oooi
`
`rtOoi
`
`OOi
`
`o.i
`
`i
`
`io
`
`,,“,,,‘,,,,,‘(cid:1)
`100
`
`1000
`
`[gelonin],
`
`nM
`
`formed
`versus
`lar HuM195
`a Cells
`
`concentration
`and rOd.
`incubated
`
`in suspension
`
`culture
`
`with
`
`immunotoxin
`
`or on-
`
`time
`
`shown
`
`and
`
`then
`
`washed
`
`and
`
`plated
`
`for
`
`dose-response
`HuMb9S-rGel
`I
`Fig.
`were
`incubated
`106/mb,
`cells
`of
`sity
`(L(cid:1)b) at
`the
`concentrations
`shown
`methylcellulose-containing
`medium.
`counted
`from four
`wells
`after
`
`replicate
`
`Starting
`of HL6O.
`with
`HuM195-rGel
`for
`72 h,
`then washed
`Colonies
`of
`>20
`5 days. Data
`are means;
`
`with
`(U)
`
`a cell den-
`or
`free
`rGel
`and
`plated
`in
`cells
`were
`SE.
`bars,
`
`Cells
`
`Freshly
`
`the
`
`and
`
`for
`
`of
`flow
`
`doses
`
`=
`
`P
`have
`can
`
`AML
`with
`Patients
`from
`Obtained
`Blast
`to HuM19S-rGel
`Sensitive
`Are
`in Vitro.
`the
`determined
`We
`using
`culture
`blasts
`in primary
`of HuMl95-rGel
`on AML
`effect
`cultures.
`and methylcellulose
`liquid
`a combination
`of
`cultures
`diagnosed
`Samples
`were
`obtained
`from
`15 patients
`with
`newly
`or
`relapsed
`AML
`and
`1 patient
`with
`chronic
`myebogenous
`blast
`leukemia
`crisis.
`The
`CD33
`expression
`measured
`by
`flow
`on
`bone
`marrow
`blasts
`ranged
`from
`75.7
`to 99.8%.
`cytometry
`for
`Incubation
`24 h in liquid
`culture
`with HuM195-rGel
`resulted
`in
`dose-responsive
`decreases
`in
`clonogenic
`cell
`recovery
`for
`patient
`each
`of
`seven
`samples
`as measured
`colony
`formation
`by
`in methylcellubose-containing
`medium.
`inhibition
`due
`Growth
`to equimolar
`concentrations
`of unconjugated
`HuMl95
`and
`rGel
`only
`was
`seen
`at very
`high
`concentrations
`100
`nM). The
`IC50
`((cid:18)
`of
`immunotoxin
`was
`1-30
`nM for
`patient
`samples
`and
`0.3
`nsi
`for
`IC50
`of
`(24-h
`incubation).
`In
`contrast,
`unconjugated
`HL6O
`each
`monoclonal
`antibody
`rGel
`(mixture
`of both molecules,
`at
`the
`stated
`concentration)
`was
`100-200
`nrvi (Table
`1). Prolong-
`ing
`the
`incubation
`time
`to 72 h had
`a variable
`effect
`on
`IC50
`but
`patient
`samples
`consistently
`lowered
`the
`IC50
`for
`unconju-
`and
`gated
`antibody
`rGel.
`samples
`Patient
`had
`bone
`marrow
`blasts
`by
`on
`with
`positive
`staining.
`cells
`presence
`of CD33
`on
`the
`accurate
`measure
`receptor
`obtained
`from
`cytometry
`to
`immunotoxin.
`Fig.
`2
`shows
`increasing
`CD33
`positivity
`Recent
`studies
`in our
`of HuM195-rGel
`up
`
`expression,
`CD33
`varying
`as
`the
`cytometry,
`flow
`Although
`this method
`cell
`surface,
`it may
`density.
`We
`asked
`would
`correlate
`the
`trend
`of
`-0.25,
`(r
`=
`laboratory4
`to
`12.5 mg/kg
`
`measured
`of
`percentage
`the
`confirms
`an
`not
`give
`whether
`results
`with
`sensitivity
`lower
`IC50
`with
`0.59).
`demonstrated
`be
`safely
`
`that
`admin-
`
`conjugated
`colony
`
`molecules
`formation.
`
`for
`
`iax
`
`T
`
`10#{128}
`
`C
`
`C
`
`it
`
`HuM195
`rGel
`
`+
`
`IIi(cid:1)
`
`3
`(95%)
`
`2
`
`(97%)
`
`4
`(88%)
`
`7
`(90%)
`
`H
`
`HL6O
`
`1
`
`(99%)
`
`II
`
`6
`
`(76%)
`
`5
`(82%)
`
`Patient
`
`Number
`
`(percent
`
`CD33-positive)
`
`patients
`from seven
`cells
`for blast
`IC50 of HuM195-rGel
`2
`Fig.
`the
`by FACS.
`To establish
`for CD33
`of cells
`positive
`percentage
`washed
`for
`24
`h and
`then
`immunotoxin
`incubated
`with
`cells
`were
`was
`estimated
`days.
`The
`IC,0
`plated;
`colonies
`were
`counted
`after
`range
`of
`IC50
`were
`linear
`in
`the
`from
`dose-response
`curves,
`which
`the
`scale. E, mean
`IC50s
`obtained
`when
`plotted
`on a log/log
`SD)
`of
`(bar,
`with
`three
`patient
`samples
`incubated
`with
`equimolar
`HuMl95
`and
`free
`(cid:1),
`IC50 for HL6O, which
`rOd.
`is 100% CD33”#{176}’.
`
`versus
`IC50,
`and
`
`5-7
`
`this
`At
`should
`
`to
`
`of up
`above
`
`istered
`12.5
`the
`
`2
`
`concentrations
`blood
`dose,
`to mice.
`iv.
`be
`safely
`attained
`and
`are well
`(70
`nM)
`p.g/mb
`blast
`cells
`from
`AML
`patients.
`for
`IC50
`concentrations
`To fur-
`Antileukemic
`Effect.
`Potency
`of HuM195-rGel
`respect
`to
`of HuMl95-rGeb
`with
`evaluate
`the
`potency
`ther
`HL6O
`and
`IC90
`empirically
`the
`for
`we
`determined
`log-kill,
`IC50
`The
`for HL6O
`shows
`a 4-log
`(data
`not
`shown).
`patient
`free
`to HuMl95-rGel
`rOd,
`in
`sensitivity
`difference
`versus
`a 2-log
`difference.
`The
`patient
`sample
`IC90
`shows
`the
`whereas
`sensitive
`to immunotoxin
`than
`to unconjugated
`anti-
`was more
`IC50
`rOe!
`by
`a factor
`of 25 at
`and
`by
`a factor
`of 9
`body
`and
`at
`the
`IC90.
`These
`results
`indicate
`the
`degree
`of
`antibody-
`
`4 M. Rosenblum,
`
`unpublished
`
`data.
`
`the
`that
`
`Downloaded from
`
`clincancerres.aacrjournals.org
`
`on October 7, 2014. © 1998 American Association for Cancer
`Research.
`
`IMMUNOGEN 2160, pg. 4
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`1974
`
`HuM195-rOel
`
`Antileukemic
`
`Activity
`
`F
`I,
`(cid:1)t
`Ii.
`(cid:1) (cid:1) (cid:1) (cid:1) (cid:1) (cid:1) (cid:1) (cid:1) (cid:1)(cid:1) (cid:1) (cid:1) (cid:1) (cid:1) (cid:1) (cid:1) (cid:1) (cid:1)(cid:1) (cid:1) (cid:1) (cid:1)-
`
`-
`
`Q\
`
`0
`
`C
`
`a
`ca
`Q(cid:1)
`
`C
`
`a
`cO
`
`-
`
`(cid:1)-
`
`((cid:1)l
`
`(cid:1)-
`
`cn
`
`(cid:1)-
`
`(cid:1)-
`
`‘.0
`
`v(cid:1)
`
`(cid:1)-
`
`90
`
`80
`
`70
`
`50
`
`40
`
`30
`
`20
`
`10
`
`.(cid:1)
`
`0
`
`4(cid:1)
`
`a(cid:1)6
`
`0
`
`1..
`
`0 = c
`
`-I
`
`aa 0a0 C
`
`-)
`
`Fig.
`(1:).
`and
`
`3
`
`Enhanced
`1 nM immunotoxin
`is expressed
`as
`
`effect
`
`combined
`immunotoxin
`of
`24 h (a),
`immunotoxin
`or
`for
`a percentage
`of
`control.
`
`with
`
`Twelve
`cryopreservation.
`followed
`by freeze/thaw
`
`patient
`(U). Mean
`
`samples,
`colony
`
`plated
`were
`all CD33(cid:1))(cid:1)s,
`number
`was determined
`
`freeze/thaw
`after
`from four
`replicate
`
`alone
`wells
`
`but
`
`across
`uniform
`not
`was
`specificity
`targeting
`was measurable
`that
`there
`for
`1-bog reduction
`in cbonogenic
`
`dose-response
`the
`at concentrations
`cells
`during
`a 24-h
`
`the
`
`by
`is
`
`blast
`
`a
`
`mediated
`range,
`necessary
`incubation.
`Has
`HuM19S-rGel
`Cryopreservation.
`with
`Synergy
`chemotherapy
`followed
`high-dose
`with
`Treatment
`of AML
`autologous
`marrow
`bone
`cryopreserved
`transplantation
`of
`presence
`of
`leukemic
`to
`due
`frequently
`unsuccessful
`autograft.
`Cryopreservation
`marrow
`bone
`progenitors
`in
`the
`to which
`other
`purging
`effect,
`itself
`has
`some
`antileukemic
`determine
`whether
`cryo-
`(14).
`To
`modalities
`have
`been
`added
`of HuMl9S-rGel
`against
`the
`effect
`preservation
`would
`enhance
`cells
`from 12 patient
`sam-
`suspended
`CD33P(cid:1)s
`blasts,
`we
`AML
`immediately
`after
`exposure
`to
`in
`ples
`medium
`freezing
`determined
`clonogenic
`cell
`recovery
`then
`HuM195-rOel
`(1 nM),
`in
`Fig.
`indicate
`that
`there
`is
`3
`after
`thawing.
`Data
`shown
`against
`but
`one
`patient
`sample.
`all
`greater
`than
`additive
`effect
`CD33
`to HuM195-rGel
`Is Reexpressed,
`and
`Sensitivity
`for
`Cells.
`Measure-
`Is Maintained
`after
`Selection
`CD33bo(cid:1)(cid:1)
`of CD33
`expression
`by flow cytometry
`reveals
`a unimodal
`ment
`or
`distribution
`within
`which
`there
`are
`individual
`cells
`with
`bower
`higher
`CD33
`expression.
`We
`asked
`whether
`cells
`that
`are
`reba-
`of
`tively
`resistant
`to HuMl95-rGel
`on
`the
`basis
`low
`CD33
`more
`expression
`would
`produce
`highly
`expressing
`cells
`on
`rise
`to
`clonal
`expansion
`give
`resistant
`clone
`stably
`with
`down-regulated
`expression.
`HL6O cells were
`separated
`
`or
`CD33
`
`a
`
`CD33-dim
`again
`
`4,
`
`FACS.
`after
`with
`
`CD33
`6 days
`mean
`
`in
`for
`
`by
`populations
`and
`CD33-bright
`into
`flow
`cytometry
`by
`expression
`was measured
`100%
`CD3Y(cid:1)S
`were
`in
`culture.
`Both
`populations
`respectively.
`166.3,
`and
`channel
`numbers
`of
`166.7
`reflected
`also
`was
`expression
`persistence
`of CD33
`The
`preincubation
`after
`the dose-response
`curves
`of OCIJAML5
`concentration.
`inhibitory
`0-6
`days
`with
`immunotoxin
`at
`an
`OCI/AML5
`immunotoxin,
`After
`or
`6 days
`exposure
`to
`2,
`that
`is
`stable,
`continued
`show
`sensitivity
`to HuMl95-rOeb
`to
`(Fig.
`4). Cells
`cells
`although
`as great
`as
`that
`seen with
`naive
`not
`only
`faint
`showed
`incubated
`with
`immunotoxin
`for
`days
`5
`5.6
`fluores-
`intensity,
`staining
`for CD33
`(median
`fluorescence
`control
`not
`the
`units
`in
`cence
`units
`196.1
`fluorescence
`versus
`a decrease
`was
`that
`there
`treated
`with
`immunotoxin).
`To confirm
`were
`then
`cells
`sites,
`in
`the
`density
`of
`immunotoxin
`binding
`anti-
`by
`FITC-labeled
`exposed
`to fresh
`immunotoxin
`followed
`median
`human
`IgG.
`Pretreated
`cells
`again
`showed
`decreased
`498
`fluo-
`fluorescence
`intensity
`(85
`fluorescence
`units
`immunotoxin
`rescence
`in naive
`cells),
`indicating
`decreased
`units
`incubation
`for
`binding.
`expression
`was
`restored
`after
`CD33
`in
`days
`immunotoxin-free
`media
`(205
`fluorescence
`units
`pretreated
`220
`fluorescence
`units
`untreated
`cells).
`in
`versus
`transient
`decrease
`in CD33
`positivity
`correlates
`with
`the
`pected
`time
`course
`for
`internalization
`of
`immunotoxin-bound
`CD33
`molecules
`and
`expression
`new
`CD33
`on
`the
`(8).
`surface
`
`versus
`
`5
`in
`The
`cx-
`
`cell
`
`of
`
`Downloaded from
`
`on October 7, 2014. © 1998 American Association for Cancerclincancerres.aacrjournals.org
`
`
`Research.
`
`IMMUNOGEN 2160, pg. 5
`Phigenix v. Immunogen
`IPR2014-00676
`
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`

`

`Clinical
`
`Cancer
`
`Research
`
`1975
`
`row cultures
`
`and
`
`SCID-Hu
`
`(severe
`
`combined
`
`immunodeficiency
`
`xenograft
`usually
`express
`
`show
`growth
`
`with
`human
`mice
`progenitors
`blast
`in S phase)
`and
`more
`differentiated
`matopoiesis.
`Raymakers
`elonal
`marker
`status
`
`of
`
`rather
`more
`
`Ml
`
`likely
`
`contrast,
`Ref.
`6).
`fraction
`proliferative
`resembling
`receptors,
`in normal
`progenitors
`reported
`the morphology
`et a!.
`grown
`from
`CD34(cid:1))(cid:1)s/CD33p(cid:1)s
`colonies
`AML.
`from
`33
`patients
`with
`blasts
`versus
`CD34P0S/CD33fleg
`from
`CD34(cid:1))(cid:1)S/CD33(cid:1)(cid:1)ee
`cells
`pre-
`patients
`whose
`blasts
`were
`rise
`CD33(cid:1)(cid:1)(cid:1)s
`dominantly
`gave
`to multilineage
`colonies
`that were
`markers
`(lacking
`diploid
`cbonal
`in five
`patients
`with
`cytogenetic
`abnormalities),
`demonstrating
`that
`these
`were
`residual
`normal
`stem
`cells
`than
`CD33(cid:1)(cid:1)Ce
`leukemic
`progenitors.
`AML
`blasts
`with
`immature
`phenotype
`(French-American-British
`expression
`classification:
`and M2)
`tend
`to have
`lower
`CD33
`and most
`can
`include
`CD33’(cid:1)5
`leukemic
`progenitors
`
`In
`
`mice;
`a high
`factor
`
`(18)
`
`AML
`(50%
`the
`he-
`and
`
`in
`
`some
`
`a!.
`
`vitro
`in
`be
`would
`CFU-leukemic
`ing
`unit-erythroid,
`
`cases
`(18).
`to that
`similar
`is
`of HuM195-rOel
`effect
`The
`antileukemic
`a CD33-directed
`(19)
`using
`by Roy
`reported
`previously
`et
`In clinical
`trials,
`intact
`ricin.
`modified,
`immunotoxin
`containing
`have
`been
`associ-
`ricin
`incorporating
`however,
`immunotoxins
`are
`often
`life-threat-
`that
`vascular
`changes
`ated with
`generalized
`due
`to the
`antibody-independent
`effects
`ening
`and most
`likely
`retains
`significant
`toxicity
`the
`blocked
`ricin,
`which
`2, 20).
`In contrast,
`free
`(1,
`cular
`endothelium
`an
`our
`results
`show
`that
`conjugation
`less
`toxic:
`yet
`toxicity
`yields
`CD33
`antibody
`an immunotoxin
`with
`reported
`that
`HL6O
`and
`primary
`AML
`blasts
`comparable
`conjugated
`for
`anti-MY9-bR
`(MY9
`monocbonal
`antibody
`progenitors
`blocked
`ricin).
`Inhibition
`of
`normal
`hematopoietie
`has
`also
`been
`demonstrated
`with
`anti-MY9-bR
`expected
`with
`HuM195-rGel.
`Direct
`comparison
`with
`CFU-granubocyte/macrophage,
`blast
`or
`CFU-granubocyte-erythrocyte-maero-
`
`rOd
`
`toward
`appears
`with
`
`selective
`with
`
`of
`vas-
`to be
`anti-
`
`to
`
`to
`
`and
`of
`form-
`
`not
`
`in
`vivo
`progen-
`
`in
`
`vivo
`
`as
`
`phage-megakaryocyte
`therapeutic
`advantage,
`itors
`respond
`differently
`Phase
`I studies
`immunogenicity
`and
`HuM195-rOel
`in
`reported
`separately
`trations
`of
`the
`
`vivo.
`in
`limited
`vivo.
`
`to
`predict
`used
`be
`should
`respective
`the
`because
`however,
`conditions.
`culture
`to cell
`toxicity
`the
`to assess
`are
`needed
`in humans
`effects
`of
`The
`of HuMl95-rOel.
`have
`been
`xenografting
`mice
`with
`HL6O
`nude
`concen-
`antileukemic
`that
`demonstrate
`and
`without
`achieved
`be
`can
`immunotoxin
`rico
`in
`as
`sensitive
`not
`were
`samples
`(2 1 ). Patient
`toxicity
`significant
`by
`concentra-
`to
`killing
`are
`nevertheless
`susceptible
`but
`HL6O
`of
`an
`immu-
`potential
`achievable
`therapeutic
`The
`tions
`is
`immunogenic
`when
`can
`if
`the molecule
`be
`notoxin
`Treatment
`with murine
`monoclonal
`anti-
`administered
`in
`followed
`the
`appearance
`of
`human
`anti-
`by
`typically
`bodies
`is
`which
`can
`neutralize
`an
`immunotoxin
`7).
`(4,
`mouse
`antibody,
`murine
`complementarity-determining
`regions
`HuM 195 contains
`of
`and
`variable
`framework
`domains
`consist
`only:
`the
`constant
`not
`human
`IgGI
`sequences.
`In
`a Phase
`I
`HuM 195
`did
`induce
`the
`formation
`of human
`anti-mouse
`antibody
`and
`could
`be
`administered
`repeatedly
`It
`remains
`to
`be
`determined
`(8).
`whether
`HuMl95-rGel
`immunogenic
`humans,
`however,
`is
`in
`because
`there may
`an immune
`response
`to rOd. We
`conclude
`be
`that HuM 195-rOd
`is active
`against
`AML,
`and
`that
`this
`in
`vitro
`immunotoxin
`merits
`further
`evaluation
`for
`potential
`use
`in the
`
`trial,
`
`0.25
`
`0.5
`
`1.0
`[gelonini,
`
`2.0
`
`nM
`
`4.0
`
`100
`
`(cid:1)(
`
`0 C8 0
`
`a S
`
`.(cid:1)
`0
`
`exposure
`of prolonged
`Effect
`4
`Fig.
`incubated
`Cells were
`to immunotoxin.
`48 h and then washed
`or 4.0 nM for
`2.0,
`at 48
`h were
`collected,
`nM immunotoxin
`different
`concentrations
`of
`immunotoxin,
`shown
`and
`I 20 h. Concentration
`curves
`are
`and
`for
`48
`h with
`unconjugated
`HuM195
`(s);
`and
`without
`pretreatment
`immunotoxin
`h (*), 96 h (X),
`with
`4 nM immunotoxin
`48
`for
`SE.
`quadruplicate
`wells
`shown:
`bars,
`
`are
`
`of OCI/AML5
`on dose-response
`at 0. 0.5,
`1.0,
`with
`immunotoxin
`4.0
`surviving
`and
`plated.
`Cells
`with
`washed,
`and
`resuspended
`at 96
`and
`this was
`repeated
`for OCIJAML5
`incubated
`rOd
`(LI):
`immunotoxin
`following
`pretreatment
`or 120 h (X). Means
`
`of
`
`blasts
`
`killed
`CD33 were
`express
`patients
`blasts
`from
`fresh
`demonstrate
`to
`sufficient
`Further
`survival.
`cell
`consistently
`not
`were
`although
`times,
`suggests
`
`that
`
`by exposure
`with
`AML,
`dose-dependent
`increases
`in anti-
`observed
`with
`nonspecific
`toxicity
`brief
`exposure
`
`to
`a
`
`to
`
`to
`
`vivo
`
`likely
`
`is
`targeted
`cells
`
`in
`
`(6)
`
`and
`
`CD34POS
`on size
`expression
`(97%
`potential
`
`a
`
`DISCUSSION
`that
`AML
`With
`HuMI95-rGel.
`was
`24-h
`exposure
`decreases
`in cbonogenic
`body-mediated
`toxicity
`more
`prolonged
`incubation
`was
`enhanced.
`This
`finding
`immunotoxin
`difference
`greatest
`the
`yield
`in
`Dose-related
`cells.
`nontargeted
`and
`between
`response
`the
`highest
`concen-
`at
`observed
`was
`clonogenic
`of
`killing
`indicate
`that AML
`results
`These
`tested.
`immunotoxin
`of
`trations
`to HuMl95-rOel
`because
`are
`sensitive
`progenitor
`cells
`blast
`colonies
`of
`forming
`(15).
`are
`capable
`the
`progenitor
`cells
`only
`to hematopoietic
`is
`toxic
`HuM 195-rOd
`immunoconjugate
`The
`cell
`surface
`occurs
`to the
`because
`binding
`CD33
`expressing
`cells
`the molecule
`by
`the
`cell.
`internalization
`of
`by
`is
`followed
`and
`events
`required
`to
`of
`internalization
`are
`number
`a small
`Only
`into
`the
`cell
`to irreversibly
`inhibit
`protein
`rOd
`enough
`transport
`to death
`of
`the
`cell
`(10,
`16). Nonhematopoi-
`leading
`synthesis,
`express
`CD33
`and
`do
`not
`bind
`the Ml95
`not
`do
`etic
`tissues
`(17).
`antibody
`monocbonal
`targeted
`some
`likely
`that
`It
`is
`important
`An
`to
`immunotoxin.
`sure
`CD331#{176}’(cid:1)or CD33(cid:1)(cid:1)
`is whether
`study
`of progenitor
`cells,
`population
`small
`in normal
`hematopoiesis,
`which
`would
`toxin. Wagner
`divided
`et
`a!.
`progenitors
`groups
`based
`into
`of
`cells
`backed
`CD33
`that
`primitive,
`quiescent
`population
`est multilineage
`proliferative
`
`a single
`survive
`cells
`arising
`from
`question
`blasts
`constitute
`AML
`there
`is evidence
`for which
`be
`resistant
`to immuno-
`human
`hematopoietic
`found
`that
`the
`fraction
`was
`phenotypically
`in G0-G1),
`with
`the
`in
`long-term
`bone
`
`expo-
`our
`a
`
`high-
`mar-
`
`Downloaded from
`
`on October 7, 2014. © 1998 American Association for Cancerclincancerres.aacrjournals.org
`
`
`Research.
`
`IMMUNOGEN 2160, pg. 6
`Phigenix v. Immunogen
`IPR2014-00676
`
`(cid:1)
`

`

`1976
`
`HuMl95-rOel
`
`Antileukemic
`
`Activity
`
`treatment
`marrow
`
`of patients
`for
`autobogous
`
`with AML
`or
`bone
`marrow
`
`for
`
`purging
`vivo
`ex
`transplantation.
`
`of bone
`
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