Vol.
`
`1.
`
`1319-1325,
`
`November
`
`1995
`
`Clinical
`
`Cancer
`
`Research
`
`1319
`
`Monocyte-mediated
`in the
`Presence
`(Anti-CD33
`
`of Acute
`Lysis
`the Bispecific
`of
`x Anti-CD64)’
`
`Myeloid
`Antibody
`
`Leukemia
`251
`X 22
`
`Cells
`
`Jian
`Division
`Pittsburgh
`Pennsylvania
`
`Jie-Hua
`Chen,
`of Hematology/Bone
`Medical
`Center
`15213
`
`Zhou,
`Marrow
`Pittsburgh
`
`and
`
`Edward
`and
`Transplantation,
`Cancer
`
`Institute,
`
`D. Ball2
`University
`Pittsburgh,
`
`of
`
`and
`anti-CD33
`humanized
`in the
`treatment
`application
`in the management
`of minimal
`
`anti-FcyRI
`of myeboid
`residual
`
`could
`leukemia,
`disease.
`
`have
`
`clinical
`especially
`
`using
`cells
`
`INTRODUCTION
`cell
`Specific
`tumor
`BsAbs3
`to direct
`immune
`novel
`immunotherapeutic
`and
`
`merous
`
`in
`
`vitro
`
`lysis
`effector
`approach
`
`be
`
`can
`cells
`has
`
`achieved
`toward
`been
`
`tumor
`the
`
`by
`
`applying
`cells.
`This
`focus
`nu-
`
`of
`
`vivo
`
`studies
`
`(1-4).
`
`Several
`
`immune-
`
`in
`
`triggering
`T-cell
`important
`
`expressed
`
`on
`
`molecules,
`receptor-associated
`roles
`in various
`
`FcyRI,
`including
`molecules,
`immune
`
`have
`responses.
`
`Fc-yRIII,
`Fc-yRII,
`shown
`been
`Fc’yRI
`
`and
`to play
`(CD64)
`is
`
`mainly
`
`on
`
`the
`
`surface
`
`of monocytes,
`
`macrophages,
`
`and
`
`neutrophils
`
`under
`
`certain
`
`clinical
`
`conditions
`
`(5-7).
`
`CD64
`
`expression
`
`can
`
`be modulated
`
`by
`
`various
`
`cytokines,
`
`primarily
`
`IFN--y
`
`(8-10).
`
`Previous
`
`studies
`
`have
`
`shown
`
`that
`
`in
`
`vito
`
`admin-
`
`istration
`
`of G-CSF
`
`increases
`
`the
`
`Fc’yRI
`
`expression
`
`phils
`
`(8),
`
`whereas
`
`administration
`
`of
`
`GM-CSF
`
`in
`
`of
`
`t’ivo
`
`in-
`
`neutro-
`can
`monocytes
`phago-
`gran-
`and
`the
`cells,
`affinity
`high
`a BsAb
`Such
`lysis
`of
`the
`expression
`on
`not
`on
`other
`cells
`(1 1-13).
`cells
`have
`(14).
`a BsAb
`was
`capa-
`cells
`against
`patients
`(15).
`
`elicit
`
`but
`
`the
`
`of
`
`under
`can
`be
`Recent
`
`of
`of
`
`cell
`
`on
`
`of
`
`ABSTRACT
`to
`(BsAb)
`antibodies
`bispecific
`Immunotherapy
`has
`cells
`target
`tumor
`toward
`effector
`immune
`direct
`Several
`of
`in a number
`studies.
`to be
`effective
`shown
`been
`Among
`have
`been
`characterized.
`trigger
`molecules
`immune
`in antibody-
`Fc’yRI
`appears
`to play
`an important
`role
`them,
`mainly
`dependent
`cellular
`cytotoxicity.
`It
`is
`expressed
`certain
`monocytes,
`macrophages,
`and
`neutrophils
`regulated
`clinical
`situations.
`The
`expression
`of Fc’yRI
`studies
`by a variety
`of cytokines,
`primarily
`by IFN-y.
`factor
`have
`shown
`that
`granubocyte-cobony-stimulating
`stimulating
`(G-CSF)
`and
`granubocyte-macrophage-cobony
`the
`FcyRI-
`factor
`(GM-CSF)
`can
`increase
`the
`number
`FcyRI
`positive
`monocytes,
`increase
`the
`expression
`and
`greatly
`circulating
`neutrophils
`after
`infusion,
`in
`vivo
`neutrophils.
`enhance
`cytotoxic
`activity
`of
`circulating
`the
`surface
`CD33
`glycoprotein
`expressed
`on
`the
`a
`and myeboid
`mature
`monocytes,
`progenitor
`cells,
`myeloid
`leukemic
`blasts,
`the
`earliest
`hematopoietic
`pro-
`not
`on
`but
`genitor
`cells
`and
`tissues.
`Herein,
`we
`the
`normal
`report
`other
`construction
`of
`22,
`conjugating
`anti-
`x
`251
`an
`a BsAb,
`CD33
`mAb
`(mAb
`to an anti-Fc-(cid:1)’RI
`mAb
`(mAb
`22). The
`251)
`x
`BsAb
`251
`22
`capable
`of
`enhancing
`the
`cytotoxicity
`of
`is
`several
`leukemia
`cell
`lines
`by cytokine-activated
`monocytes.
`Our
`data
`also
`show
`that
`G-CSF-
`and GM-CSF-stimulated
`cells
`monocytes
`can mediate
`cytotoxicity
`of
`target
`leukemia
`The
`comparable
`to
`that
`IFN--y-stimulated
`monocytes.
`of
`incu-
`expression
`of Fc-yRI
`on monocytes
`after
`24-h
`in vitro
`with
`bation
`G-CSF
`and GM-CSF
`was
`increased,
`although
`significantly.
`not
`Prolonged
`incubation
`of monocytes
`with
`G-CSF
`for
`48
`h significantly
`increased
`the
`FcyRI
`expres-
`humanized
`anti-CD33
`anti-Fc-(cid:1)iRI
`mAb
`and
`sion.
`Because
`are
`available,
`and
`because
`GM-CSF
`and G-CSF
`have
`been
`after
`used
`widely
`for patients
`chemotherapy
`to stimulate
`the
`recovery
`of myeboid
`hematopoiesis,
`additional
`clinical
`deveb-
`opment
`of
`this
`project
`is
`feasible.
`A BsAb
`comprised
`of
`
`is
`
`by
`
`crease
`(10).
`cytosis
`
`the
`GM-CSF
`(6,
`8),
`
`ubocytes
`BsAb
`and
`could
`target
`myeloid
`normal
`Clinical
`shown
`
`no
`
`FcyRI-positive
`circulating
`of
`number
`neutrophib
`to stimulate
`shown
`been
`has
`ADCC
`by monocytes
`to enhance
`and
`as
`immune
`effector
`use monocytes
`To
`(5-7).
`to Fc-yRI
`with
`capable
`of binding
`be
`should
`cell
`surface.
`antigen
`on
`a tumor
`to a specific
`cells
`and
`direct
`monocytes
`toward
`tumor
`with
`restricted
`cells.
`CD33
`is a glycoprotein
`blasts,
`progenitor
`cells
`and
`leukemic
`progenitor
`tissue
`and
`early
`hematopoietic
`on
`leukemia
`trials
`targeting
`mAb
`to CD33
`body
`tissues
`significant
`toxicity
`to
`normal
`synthesis
`have
`reported
`previously
`We
`25 1 X 3G8
`This
`BsAb
`(anti-CD33
`X anti-FcyRIII).
`of natural
`killer
`ble
`of
`augmenting
`cytotoxicity
`the
`fresh
`cells
`from
`CD33-positive
`cell
`lines
`and
`AMI
`In this
`study,
`we
`describe
`a new BsAb
`251
`anti-Fc-yRI)
`that
`can
`significantly
`increase
`
`X 22
`the
`
`(anti-CD33
`cytotoxicity
`
`X
`of
`
`CD33-positive
`GM-CSF)-activated
`
`leukemia
`
`cells
`monocytes.
`
`by
`
`cytokine
`
`(IFN-y,
`
`G-CSF,
`
`and
`
`MATERIALS
`mAbs.
`Purified
`
`(i6).
`
`AND METHODS
`251
`(anti-CD33)
`and
`the
`anti-FcyRI
`
`mAb
`251
`
`was
`
`developed
`mAb
`(mAb
`
`by E. D. B.
`22)
`were
`
`used
`
`are: BsAb,
`abbreviations
`3 The
`colony-stimulating
`granubocyte-macrophage
`Sulfo-SMCC,
`imidyb-S-acetylthioacetate;
`imidomethyl)
`cyclohexane-L-carboxylate;
`myeboid
`stimulating
`factor;
`AML,
`acute
`fluorescence
`intensity;
`ADCC,
`antibody-dependent
`
`antibody;
`SATA,
`
`bispecific
`factor;
`sulfosuccinimidyl
`G-CSF,
`granubocyte
`leukemia;
`cellular
`
`GM-CSF,
`N-succin-
`4 (N-male-
`colony-
`mean
`MFI,
`cytotoxicity.
`
`accepted
`6/13/95;
`revised
`1/1 1/95;
`Received
`by Grant
`in part
`supported
`was
`work
`I This
`Science
`the Blood
`and
`by
`Institute
`Cancer
`should
`reprints
`2 To whom requests
`for
`Hematology/Bone
`Marrow
`Transplantation,
`Medical
`Center,
`Lothrop
`Street,
`Pittsburgh,
`648-6413;
`Fax:
`648-6393.
`
`200
`(412)
`
`7/5/95.
`CA31888
`Foundation
`be addressed,
`University
`PA 15213.
`
`from the National
`(Pittsburgh,
`PA).
`at
`the Division
`of
`of
`Pittsburgh
`Phone:
`(412)
`
`
`
`Downloaded from on August 31, 2015. © 1995 American Association for Cancerclincancerres.aacrjournals.org
`
`
`Research.
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1014 Page 1 of 8
`
`

`
`1320 AML Cell Lysis
`
`in
`
`the
`
`Presence
`
`of BsAb
`
`251
`
`X 22
`
`Inc.
`an
`
`x 22.
`obtained
`was
`SATA
`
`cell
`
`were
`
`Lanotte
`1640
`
`obtained
`MD).
`The
`(Paris,
`medium
`(100
`units/
`Grand
`Island,
`5% CO2. All
`cell
`contamination
`San Diego,
`for CD33,
`
`less
`
`con-
`1% CD3-
`Then,
`the
`
`a
`
`a
`
`at
`was
`
`DC
`The
`
`Assay
`of
`the
`
`are
`both
`NJ);
`(Annandale,
`by Medarex,
`provided
`kindly
`directed
`mAb,
`IgG2a
`murine
`mAbs.
`SCCL-1,
`IgGi
`murine
`this
`study
`as
`was
`used
`in
`tnansfernin
`receptor,
`the
`against
`mAb
`(17).
`an
`unconjugated
`via
`control
`for
`ADCC
`positive
`digestion
`prepared
`by pepsin
`fragment
`of mAb
`251 was
`F(ab’)2
`and
`purified
`using
`a protein
`A column
`(Pharmacia
`Biotech,
`Inc.,
`Piscataway,
`NJ).
`251
`of BsAb
`chemicals
`Conjugation
`Production
`Chemical
`from Pierce
`were
`and
`Sulfo-SMCC
`SATA
`from Sigma
`purchased
`Co.
`(Rocklord,
`IL). Hydroxylamine
`(SO mM)
`Chemical
`Co.
`(St.
`Louis,
`MO).
`was
`freshly
`and
`Sulfo-SMCC
`(50
`prepared
`in
`100%
`dimethylformamide
`in PBS
`mAb
`251
`with
`in
`mixed
`mM),
`(pH
`7.4).
`SATA
`was
`room
`temperature,
`at
`i-h
`final
`molar
`ratio
`10:1.
`After
`reaction
`nonneacted
`SATA
`was
`removed
`by
`size-exclusion
`centnifuga-
`tion
`through
`a Centnicon
`30
`apparatus
`(Amicon,
`Inc.,
`Beverly,
`free
`MA).
`The
`sulfhydryl
`group
`was
`generated
`by deacetylation
`with
`hydroxylamine
`at
`room
`temperature
`for
`Excess
`hy-
`2
`h.
`droxylamine
`was
`removed
`by
`a Centricon
`30
`apparatus.
`the
`At
`same
`time,
`Sulfo-SMCC
`was
`reacted
`with
`the
`mAb
`22
`in
`a final
`molar
`ratio
`of 20: 1 at
`room
`temperature
`for
`2 h. The maleimide-
`by
`activated
`mAb22
`was
`separated
`from unreacted
`Sulfo-SMCC
`con-
`30
`apparatus.
`The
`final
`centrifugation
`through
`a Centnicon
`out
`jugation
`carried
`by mixing
`equal
`molar
`amounts
`of both
`was
`mAbs
`room
`temperature
`overnight.
`The
`concentration
`of
`BsAbs
`determined
`a Bio-Rad
`Protein
`using
`(Bio-Rad
`Laboratories,
`Richmond,
`CA).
`purity
`BsAbs
`verified
`SDS-PAGE.
`was
`by
`Cell Lines.
`lines
`and KG-la
`HL-60
`(Rockville,
`from American
`Type
`Culture
`Collection
`NB4
`cell
`line
`was
`kindly
`provided
`by Dr. M.
`France).
`Cell
`lines
`were
`maintained
`in RPM!
`containing
`10% FCS,
`L-glutamine
`penicillin
`(2 mM),
`ml),
`and
`streptomycin
`(SO ji.g/ml)
`(GIBCO-BRL,
`NY)
`at 37#{176}Cin a humidified
`atmosphere
`with
`lines
`were
`checked
`periodically
`for Mycoplasma
`using
`the DNA
`hybridization
`method
`(Gen-Probe,
`CA).
`HL-60
`and NB4
`cells
`are
`strongly
`positive
`are
`whereas
`KG-la
`cells
`negative
`for CD33.
`freshly
`were
`of Effector
`Cells.
`Monocytes
`Preparation
`after
`they
`donors
`separated
`from
`peripheral
`blood
`of normal
`the
`(Pharmacia,
`Pis-
`gave
`informed
`consent.
`After
`Ficoll-Hypaque
`mononuclear
`cells
`cataway,
`NJ)
`gradient
`centnifugation,
`the
`were
`collected
`and washed
`twice.
`The
`cells were
`incubated
`with
`mAb
`(American
`Type
`Culture
`Collection)
`and mAb
`6G7
`OKT3
`twice.
`(anti-CD45RA)4
`for
`1 h
`at
`4#{176}Cand
`washed
`Dynabeads
`M-4S0
`coated
`sheep
`antimouse
`IgG (DYNAL,
`Great
`Neck,
`with
`NY) were
`added
`at a final
`ratio
`of 20:1
`and
`incubated
`at 4#{176}Cfor
`1 h. The
`lymphocytes
`were
`depleted
`using
`a handheld
`magnet.
`The
`procedure
`was
`repeated
`twice.
`purity
`of monocytes
`was
`The
`analyzed
`by
`flow
`cytometry,
`and
`the morphology
`of
`the
`cells
`was
`verified
`by microscopic
`examination
`of Wright-Giemsa-
`stained
`cytospin
`preparations.
`The
`separated
`monocytes
`tamed
`more
`than
`95% CDT4-positive
`cells,
`than
`positive
`cells,
`and
`less
`than
`1% CDS6-positive
`cells.
`
`were
`
`the
`
`(Genentech,
`Seattle,
`WA),
`the
`cytotox-
`cytokines
`
`pur-
`CA).
`by
`
`In
`
`a
`a
`
`X
`
`22
`
`fragment
`
`IFN-(cid:1)y
`units/ml
`200
`with
`incubated
`monocytes
`(Immunex,
`G-CSF
`CA),
`10 ngjml
`San
`Francisco,
`24
`h before
`for
`GM-CSF
`(Immunex)
`or
`10 ng/ml
`in medium
`without
`Monocytes
`incubated
`icity
`assay.
`control.
`in each
`assay
`as
`were
`included
`or
`FITC-
`Analysis.
`phycoerythnin-
`Cytometric
`Flow
`were
`and
`anti-CD56
`anti-CD13,
`anti-CD3,
`conjugated
`Jose,
`Immunosystem
`(San
`Becton
`Dickinson
`from
`chased
`(anti-Fc-yRI)
`provided
`was
`mAb
`FITC-conjugated
`22
`expres-
`For
`evaluation
`of Fc’yRI
`(Annandale,
`NJ).
`Medarax,
`Inc.
`were
`di-
`before
`after
`cytokine
`incubation
`and
`sion,
`monocytes
`of
`im-
`For
`with
`this
`of mAbs.
`evaluation
`panel
`rectly
`stained
`HL-
`from
`binding,
`monocytes,
`as well
`as
`cells
`munoconjugate
`were
`1 p.g/ml
`and NB4
`lines,
`incubated
`with
`cell
`60, KG-la,
`cells
`twice
`and
`X 22
`for
`1 h. The
`were
`washed
`BsAb
`251
`goat
`with
`FITC-conjugated
`F(ab’)2
`anti-mouse
`IgG
`incubated
`Francisco,
`All
`for
`(Caltag,
`South
`San
`CA)
`30 mm.
`of
`(H + L)
`by
`FACScan
`were
`analyzed
`(Becton,
`Dickinson).
`the
`samples
`phycoerythrin-conjugated
`mouse
`isotypic
`immuno-
`FITC-
`or
`were
`used
`as
`controls.
`globulins
`performed
`was
`assay
`Cytotoxicity
`Assay.
`The
`96-well
`in
`Co.,
`Instrument
`(Rainin
`round-bottomed
`micnotiter
`plates
`RPMI
`once
`with
`washed
`Woburn,
`MA).
`The
`target
`cells
`were
`chro-
`sodium
`[51Cr]
`p,Ci
`1640 medium
`and
`incubated
`with
`100
`for
`1 h at 37#{176}C.After
`MA)
`mate
`(New
`England
`Nuclear,
`Boston,
`resuspended
`in RPM!
`being
`washed
`several
`times,
`the
`cells were
`a concentration
`of
`1 X
`1640 medium
`containing
`10% FCS
`to
`cells
`were
`suspended
`in
`105/ml.
`Monocytes
`serving
`as
`effector
`RPMI
`1640 medium
`to
`a final
`concentration
`of
`1 X
`107/ml.
`Effector
`cells
`(100
`(cid:1)i.l) were
`added
`to the
`first
`row of
`cells,
`and
`a serial
`dilution
`performed
`with
`equal
`volumes
`of RPM!
`was
`1640 medium.
`100
`p.1 target
`cells were
`added
`in the wells
`Then,
`to yield
`final
`ratios
`of 50:1,
`25:1,
`and
`12:1.
`a standard
`E:T
`assay,
`0.2
`(cid:1)ig
`immunoconjugate
`was
`finally
`added
`to
`yield
`final
`immunoconjugate
`concentration
`of
`1 p.g/ml.
`SCCL-l,
`and
`mouse
`IgG2a
`mAb
`directed
`against
`transfernin
`receptor
`capable
`of binding
`to the Fc’yRI
`through
`its Fe domain
`(17), was
`included
`in
`each
`assay
`as
`a
`positive
`control
`to measure
`the
`of
`activity
`the monocytes.
`Several
`other
`controls
`with
`E:T ratios
`of 50: 1 also were
`incorporated
`in each
`assay,
`including
`incuba-
`tion
`of
`target
`cells
`with
`the
`immunoconjugate
`alone,
`incubation
`of
`target
`and
`effector
`cells with
`no antibody,
`incubation
`of
`target
`and
`effector
`cells with
`the unconjugated
`parental
`antibodies,
`and
`incubation
`of
`effector
`cells
`with
`CD33-negative
`leukemia
`cells
`in
`the
`presence
`of BsAb
`251
`In
`each
`assay,
`a 20-fold
`22.
`excess
`of
`unconjugated
`mAb
`or mAb
`251
`along
`with
`the
`immunoconjugate
`were
`incubated
`together
`with
`the
`target
`and
`to
`effecton
`cells
`determine
`whether
`tumor
`cell
`lysis
`could
`be
`blocked
`by either
`of
`the
`parental
`antibodies.
`Cytotoxicity
`assays
`also were
`performed
`to
`compare
`the
`cytotoxicity
`mediated
`by
`x 22, mAb
`the BsAb
`251
`of mAb
`251,
`and
`F(ab’)2
`whether
`determine
`To
`251
`at
`various
`E:T
`ratios.
`presence
`of
`the
`with
`interfere
`could
`human
`immunoglobulin
`redirected
`tu-
`the
`assays
`cytotoxicity
`cell
`mon
`killing,
`some
`performed
`in
`were
`After
`incubation
`100% human
`AB serum.
`at 37#{176}Cfor
`4 or 18 h,
`the
`the microplates
`were
`centrifuged,
`and
`supernatant
`was
`col-
`lected
`for
`the
`determination
`of 51Cr
`release.
`Maximum
`lysis was
`achieved
`by
`the
`addition
`100
`p.1 5%
`NP4O
`to
`p.1 target
`cells.
`percentage
`lysis
`was
`calculated
`(experi-
`
`4 J. H. Zhou,
`
`unpublished
`
`observations.
`
`The
`
`of
`cell
`
`of
`
`100
`as:
`
`
`
`Downloaded from on August 31, 2015. © 1995 American Association for Cancerclincancerres.aacrjournals.org
`
`
`Research.
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1014 Page 2 of 8
`
`

`
`Clinical
`
`Cancer
`
`Research
`
`1321
`
`Table
`
`I
`
`Th e binding
`
`of mAb
`
`22, m Ab
`
`251,
`
`and
`
`Bs Ab
`
`251
`
`X 22
`
`to target
`
`an d effector
`
`cells”
`
`mAb
`
`251
`
`mAb
`
`22
`
`BsAb
`
`251
`
`X
`
`22
`
`HL-60
`NB4
`KG-la
`Monocyte
`
`% Positive
`
`99.5
`89.2
`Negative
`81.6
`
`MFI
`
`126.4
`54.6
`Negative
`27.0
`
`% Positive
`
`64.6
`54.2
`Negative
`79.0
`
`MFI
`
`16.6
`12.9
`Negative
`43.9
`
`% Positive
`
`99.6
`89.1
`Negative
`94.0
`
`MFI
`
`151.0
`56.7
`Negative
`101.3
`
`“ Flow cytometric
`FITC-conjugated
`
`by indirect
`analysis
`goat
`F(ab’),
`antimouse
`
`251 (0.5
`of cells with mAb
`staining
`IgG (H + L).
`Isotype-matched
`
`3G8
`ig,’ml), mAb
`murine mAb
`controls
`
`X 22 (1
`251
`or BsAb
`gig/mI),
`(0.5
`were
`included
`in all analyses.
`
`with
`
`(cid:1).tg/mb),
`
`then
`
`.
`
`.-.0---.---
`
`NB4
`
`HL-60
`
`1oo(cid:1)
`
`80
`
`60(cid:1)
`
`40’
`
`20’
`
`‘I
`1.
`
`0Ba
`
`I-
`
`Table
`
`2
`
`Effect
`
`of
`
`cytokine
`
`stimulation
`on monocytes”
`
`on
`
`the
`
`expression
`
`of Fc-yRI
`
`Cytokine
`
`Positivity
`
`(%)
`
`MFI
`
`No. of experiments
`
`cytokine
`No
`IFN--y
`G-CSF
`GM-CSF
`
`84 ± 3
`87 ± 9
`88±6
`85 ±
`
`11
`
`54 ± 3
`150
`± 33”
`72±18
`70 ± 28
`
`3
`3
`
`3
`3
`
`of Fc-yRI
`expression
`22. Monocytes
`mAb
`incubated
`with
`
`and
`
`by direct
`were
`indicated
`
`the
`of
`analysis
`with
`FITC-conjugated
`peripheral
`blood
`the
`analysis.
`(P <
`0.05)
`
`compared
`
`to that with
`
`no cytokine
`
`I
`
`(‘ Flow cytometric
`of monocytes
`staining
`from
`normal
`separated
`24 h before
`for
`cytokine
`b Significant
`increase
`stimulation.
`
`mental
`imum
`
`cpm
`release
`
`-
`
`spontaneous
`mean
`cpm
`
`-
`
`release
`spontaneous
`
`mean
`
`cpm)
`mean
`
`(max-
`/
`X 100
`cpm).
`Spontane-
`
`cells
`less
`
`25
`
`release
`ous
`maximum
`assay.
`diluted
`
`For
`and
`Statistical
`totoxicity
`assays
`mean
`SD.
`The
`±
`paired
`Student’s
`
`t
`
`from
`51Cr
`of
`in a 4-h
`release
`dose-response
`the E:T
`added;
`Analysis.
`was
`obtained
`significance
`test
`when
`
`of
`than
`less
`was
`target
`the
`5%
`18-h
`15% in an
`than
`and
`assay
`1 X 22
`was
`serially
`BsAb
`assays,
`assays
`was
`50:1.
`in those
`ratio
`experimental
`result
`Each
`in triplicate
`reported
`and
`level
`was
`determined
`applicable.
`
`in
`
`the
`as
`using
`
`cy-
`the
`the
`
`RESULTS
`Binding
`binding
`
`CD33
`for
`
`of BsAb
`of
`the
`two
`The
`x 22 to
`BsAb
`effector
`target
`251
`and NB4
`HL-60
`analysis.
`cytometric
`positive
`for
`weakly
`and
`tive
`for
`and
`Fc’yRI.
`The
`CD33
`both
`negative
`and monocytes
`with
`NB4,
`to HL-60,
`intensities,
`suggesting
`mean
`fluorescence
`did
`not
`alter
`the
`chemical
`conjugation
`The
`results
`of
`flow cytometric
`analysis
`x 22 could
`The
`BsAb
`251
`be detected
`HL-60
`cells
`after
`4-h
`incubation,
`
`x
`251
`parental
`
`and Effector
`22 to Target
`mAbs
`(mAb
`22
`and
`
`Cells.
`and
`
`251)
`
`and
`
`by
`
`cells
`cells
`Fc-(cid:1)RI.
`BsAb
`similar
`
`flow
`studied
`was
`posi-
`strongly
`were
`were
`cells
`KG-la
`251 X 22 bound
`percentages
`and
`the
`process
`of
`that
`characteristics.
`binding
`1.
`are
`presented
`in Table
`on
`the
`surface
`of 90% of
`suggesting
`that
`the
`BsAb
`
`not
`is
`X 22
`251
`on HL-60
`molecules
`may
`be
`modulation
`effector
`cells
`immune
`of
`target
`cytotoxicity
`to KG-ia
`not
`bind
`in the
`cytotoxicity
`as
`well
`as
`to
`cytotoxicity.
`
`CD33
`to
`binding
`after
`internalized
`readily
`antigenic
`of
`absence
`The
`surface
`(18).
`cell
`251
`BsAb
`important
`for
`to
`direct
`22
`X
`subsequent
`to
`induce
`and
`to
`target
`cells
`22
`does
`BsAb
`251
`that
`cells.
`The
`fact
`X
`controls
`as
`these
`cells
`cells
`allows
`us
`to use
`cell
`lysis,
`assay
`to measure
`nonspecific
`tumor
`validate
`the
`specificity
`of
`BsAb-mediated
`
`the
`
`I
`
`0
`
`I
`
`I
`
`I
`
`0.0001
`0.00001
`Concentrations
`
`0.001
`of BsAb
`
`I
`
`0.01
`
`251
`
`I
`
`0.1
`x 22
`
`1
`(jig/mI)
`
`10
`
`Cytotoxicity
`I
`Fig.
`of
`various
`presence
`of
`target
`increase
`25 1 X 22. Bars,
`
`cell
`SE.
`
`cells
`target
`of
`concentrations
`lysis
`was
`
`by
`
`in the
`monocytes
`IFN-y-activated
`25 1 X 22. A significant
`of BsAb
`observed
`with
`0.0()l-I()
`pg/mb
`BsAb
`
`Cells
`of Effector
`Activation
`incubation
`that
`the
`documented
`well
`of Fc-yRI
`expression
`increases
`the
`activity.
`Several
`bates
`their
`cytotoxic
`also
`that G-CSF
`and GM-CSF
`can
`FcyRI
`on
`circulating
`monocytes
`administration.
`Both
`of
`the
`cytokines
`hance
`the
`cytotoxocity
`of monocytes
`incubation,
`although
`the
`Fc’yRI
`vitro
`
`on
`
`by Cytokines.
`been
`IFN--y
`of monocytes
`stimu-
`their
`surface
`indicate
`previous
`studies
`stimulate
`the
`expression
`and
`neutrophils
`after
`in
`have
`been
`shown
`and
`neutrophils
`expression
`does
`
`It has
`with
`and
`
`of
`vivo
`
`to
`after
`not
`
`en-
`
`in
`in-
`
`the
`
`=
`
`(MFI
`
`on
`
`h
`
`crease
`
`CSF
`
`significantly.
`incubation
`of monocytes.
`tivity
`IFN--y
`for
`of
`expression,
`increase
`tion
`sion
`
`whereas
`
`251
`
`for
`required
`assay
`typical
`cell
`tumor
`251
`BsAB
`concentrations
`tive
`in
`vivo
`
`may
`
`be
`
`low
`
`effect
`studied
`We
`of Fc-yRI
`expression
`the
`of monocytes
`Incubation
`a significant
`in
`resulted
`24
`and
`GM-CSF
`G-CSF
`whereas
`(Table
`2). With
`Fc-yRI
`significantly
`48
`h,
`IFN-y
`and G-CSF
`both
`increased
`for
`and
`I 10
`±
`further
`(MFI
`218
`±
`82
`±
`16).
`64
`not
`did
`GM-CSF
`=
`the
`Presence
`in
`Cells
`of Target
`Cytotoxicity
`of BsAb
`dependence
`concentration
`22.
`The
`x
`The
`results
`measured.
`was
`cytotoxicity
`in Fig.
`1. A significant
`are
`presented
`from
`0.001
`p.g/ml
`lysis
`was
`observed
`x
`cytotoxicity
`of
`22.
`The
`presence
`of BsAb
`251
`X 22 may
`be
`important
`immunotherapy,
`because
`the
`concentration
`in
`tissues.
`
`GM-
`ac-
`
`of
`
`and
`of G-CSF
`cytotoxic
`and
`200
`units/ml
`with
`Fc-yRI
`increase
`did
`not
`incubation
`incuba-
`prolonged
`expres-
`Fc-yRI
`respectively),
`
`33,
`
`of BsAb
`251
`X 22
`
`one
`of
`in
`increase
`10
`p.g/mb
`very
`low
`for
`effec-
`of BsAb
`
`to
`at
`
`
`
`Downloaded from on August 31, 2015. © 1995 American Association for Cancerclincancerres.aacrjournals.org
`
`
`Research.
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1014 Page 3 of 8
`
`

`
`1322 AML
`
`Cell Lysis
`
`in the Presence
`
`of BsAb
`
`251 X 22
`
`A. NB4
`
`cells
`
`B. HL-60
`
`cells
`
`A. NB4
`
`cells
`
`B. HL-60
`
`cells
`
`100
`
`80
`
`60
`
`[1 40
`
`.......
`
`T
`
`2:j1]J’(cid:1)]
`
`50:1
`
`-0--
`
`-4-
`
`----0-S--
`
`--6--
`
`---w---
`
`Donor
`
`1
`
`Donor 2
`
`rs,nor
`
`Donor
`
`3
`
`4
`
`Donor 5
`
`100
`
`80
`
`(cid:1):
`,l(cid:1)-a
`
`.(cid:1)60
`
`=c(cid:1)
`
`(cid:1)40
`
`(cid:1)2:J(cid:1)J(cid:1)(cid:1)
`
`#{149}4hassay
`
`18h assay
`
`,(cid:1)
`
`100
`
`80
`
`100
`
`80
`
`60’
`
`20(cid:1)
`
`‘I,
`±)
`
`(cid:1)1
`I-
`
`0Ba
`
`I-
`
`0(cid:1) (cid:1) (cid:1)
`12:1
`25:1
`cell
`E:T
`
`#{149} 0’
`
`50:1
`
`ratio
`
`,
`25:1
`12:1
`E:T cell
`
`50:1
`ratio
`
`Cytotoxicity
`2
`Fig.
`donors.
`The
`five
`individual
`donors
`
`target
`of
`cytotoxicity
`and varies
`
`monocytes
`IFN--y-activated
`by
`cells
`the E:T
`cell
`is dependent
`on
`greatly
`among
`different
`donors.
`
`from
`ratio
`Bars,
`
`in
`SE.
`
`3
`Fig.
`monocytes
`observed
`
`12:1
`
`25:1
`
`50:1
`
`12:1
`
`25:1
`
`E:T cell
`
`ratio
`
`E:T cell
`
`ratio
`
`of
`
`Effect
`from
`18-h
`
`in
`
`cell
`on target
`time
`incubation
`donor
`2. A significant
`increase
`SE.
`assays.
`Bars,
`
`lysis
`of
`
`by IFN-(cid:1)y-activated
`target
`cell
`lysis
`
`was
`
`parental
`either
`excess
`a 20-fold
`of
`addition
`the
`4,
`of
`cytotoxicity
`increment
`inhibited
`the
`significantly
`the
`presence
`expected,
`X 22. As
`251
`by BsAb
`medi-
`cytotoxicity
`with
`the
`interfere
`did
`not
`AIB serum
`22
`of mAb
`the
`binding
`because
`X 22
`by BsAb
`251
`The
`cytotoxicity
`binding
`site.
`Fe
`the
`occurs
`outside
`was
`blocked
`(an IgG2a
`mAb)
`SCCL-l
`by mAb
`sig-
`pres-
`because
`the
`by
`human
`AB serum,
`presumably
`the
`Fe
`with
`compete
`serum
`immunoglobulin
`could
`(data
`to Fe-yRI
`mAb
`binding
`the
`IgG2a
`SCCL-1
`for
`of
`the
`effects
`the
`control
`additional
`compared
`to BsAb
`251 X 22
`its F(ab’)2
`and
`fragments
`(Fig.
`4). The
`cyto-
`leukemia
`of
`target
`cells
`its
`by mAb
`251
`and
`F(ab’)2
`fragments
`was
`that mediated
`by monocytes
`alone,
`whereas
`significantly
`enhanced
`cytotoxicity
`
`of
`
`of
`
`to
`
`the
`
`of
`
`in Table
`antibody
`mediated
`human
`ated
`Fc’yRI
`mediated
`nificantly
`ence
`of
`of
`portion
`An
`shown).
`not
`parental
`mAb
`251
`on
`the
`cytotoxicity
`toxicity
`mediated
`comparable
`with
`BsAb
`251
`X
`leukemia
`targets.
`
`22
`
`IFN-’y-
`by
`cells
`NB4
`and
`of HL-60
`lysis
`specific
`The
`cytotoxicity
`2. The
`in Fig.
`summarized
`is
`monocytes
`activated
`E:T
`At
`an
`time.
`incubation
`ratios
`and
`was
`dependent
`on E:T
`lysed
`in the
`were
`target
`cells
`70% of
`ratio
`of
`50:1,
`more
`than
`cytotoxicity
`among
`22. Variations
`of
`presence
`of BsAb
`251
`X
`target
`differences
`in susceptibility
`of
`cells
`individual
`donors
`and
`As
`shown
`in Fig.
`2, monocytes
`from
`to
`lysis
`were
`observed.
`cytotoxicity
`toward
`both
`HL-60
`and
`donor
`S had
`the
`highest
`NB4
`cells,
`whereas
`monocytes
`from donor
`3 had
`a much
`higher
`cytotoxicity
`toward
`NB4
`cells
`(40-75%)
`than
`did HL-60
`cells
`The
`expression
`of Fc-yRI
`on monocytes
`after
`IFN--y
`(25-45%).
`incubation
`was
`comparable
`in
`all
`cytotoxicity
`assays,
`and
`the
`BsAb
`25 1 X
`used
`these
`assays
`was
`derived
`from
`one
`in
`22
`The
`conjugation.
`effect
`incubation
`time
`on
`the
`cytotoxicity
`of
`important.
`assay
`was
`A comparison
`cytotoxicity
`from
`a 4-h
`of
`assay
`an
`and
`18-h
`is presented
`3.
`In
`these
`assays,
`the
`in Fig.
`leak
`background
`51Cr
`was
`of
`the maximal
`less
`than
`5%
`the
`4-h
`in
`release
`assay
`and
`15%
`of
`the maximal
`less
`than
`in the
`18-h
`release
`assay.
`The
`of
`tumor
`cell
`lysis was
`percentage
`significantly
`increased
`after
`incubation.
`This
`observa-
`an
`18-h
`is
`the
`tion,
`combined
`with
`fact
`that
`a higher
`E:T
`ratio
`required
`for
`cytotoxicity,
`suggests
`that
`the
`production
`cytotoxic
`cyto-
`kines
`may
`be
`involved
`in target
`cell
`lysis.
`on Cytotoxicity
`The
`Effect
`of Cytokine
`Activation
`Cells.
`A series
`of
`assays
`Target
`performed
`to
`compare
`was
`enhancement
`of
`cyto-
`of
`cytokine
`incubation
`on
`the
`the
`effects
`target
`cells.
`In general,
`the
`cytotoxicity
`of HL-60
`and
`of
`toxicity
`by monocytes
`incubated
`with
`G-CSF
`and GM-CSF
`cells
`NB4
`comparable
`to
`mediated
`by monocytes
`incubated
`was
`All
`!FN-y.
`three
`cytokines
`significantly
`increased
`with
`potency
`cytotoxic
`of monocytes.
`The
`results
`summarized
`3.
`in Table
`by BsAb
`Mediated
`of Cytotoxicity
`Specificity
`induced
`lysis
`was
`cell
`whether
`target
`determine
`To
`X 22, CD33-negative
`of BsAb
`251
`by the
`presence
`target
`cells
`in some
`cytotoxicity
`were
`used
`as
`in the
`presence
`of BsAb
`251
`KG-la
`cells
`(without
`ranging
`from
`3.7%
`±
`1.2%
`(with
`IFN-’y
`incubation).
`7.7%
`1.0%
`±
`be
`shown
`further
`by blocking
`cell
`can
`lysis
`excess
`concentrations
`of
`either
`parental
`
`of
`
`are
`
`of
`
`the
`
`22.
`
`x
`251
`specifically
`KG-la
`cells
`assays.
`Lysis
`of
`X 22 was
`very
`low,
`IFN-y
`incubation)
`to
`target
`The
`specificity
`of
`with
`the
`cytotoxicity
`antibody.
`As
`illustrated
`
`of
`
`that
`
`DISCUSSION
`signif-
`improved
`been
`of AML
`treatment
`the
`Although
`regimens,
`and
`agents
`new chemotherapeutic
`by
`various
`disease
`residual
`of minimal
`of
`the
`persistanee
`as
`a result
`patients.
`these
`for
`problem
`remains
`a major
`chemotherapy
`have
`transplantation
`marrow
`allogeneic
`bone
`such
`as
`current
`the
`outcomes
`of
`the
`further
`improve
`made
`to
`bone
`marrow
`transplantation
`In
`addition,
`autologous
`therapy
`for
`patients
`with
`AML.
`used
`as
`consolidation
`in
`bone
`marrow
`transplants
`of
`experiments
`residual
`leukemic
`cells
`suggested
`the
`(18).
`the
`relapse
`of
`leukemia
`can
`contribute
`including
`have
`been
`developed,
`purging
`techniques
`mAb
`plus
`with
`cytotoxic
`drugs,
`of
`marrow
`(19).
`All
`immunomagnetic
`bead
`separation
`success.
`of
`have
`met
`some
`measure
`relapse
`be-
`numbers
`of
`patients
`still
`the
`primary
`therapy
`to eliminate
`all
`to purge
`all malignant
`cells
`from
`the
`
`has
`
`that
`
`to
`
`with
`
`of
`
`in
`
`bone
`and
`strategies
`considerable
`either
`failure
`or
`failure
`vivo
`
`icantly
`relapse
`after
`Efforts
`been
`therapy.
`has
`been
`Gene-marking
`AML
`have
`transplant
`Various
`treatment
`complement,
`of
`these
`However,
`cause
`disease
`autograft.
`In this
`residual
`
`of
`
`in
`
`mate
`
`we
`study,
`leukemia
`
`examined
`cells
`
`by
`
`alternative
`an
`directing
`host
`
`strategy
`immune
`
`to elim-
`effeetor
`
`
`
`Downloaded from on August 31, 2015. © 1995 American Association for Cancerclincancerres.aacrjournals.org
`
`
`Research.
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1014 Page 4 of 8
`
`(cid:1)
`(cid:1)
`(cid:1)
`

`
`Clinical
`
`Cancer
`
`Research
`
`1323
`
`E:T
`
`cells
`NB4
`12:1
`
`25:1
`
`50:1
`
`cells
`
`HL-60
`12:1
`
`25:1
`
`50:1
`
`Table
`
`3
`
`Effect
`
`of
`
`cytokine
`
`incubation
`
`on monocyte-mediated
`
`cytotoxicity
`
`in the
`
`presence
`
`of BsAb
`
`251
`
`X 22”
`
`Donor
`
`IFN--y
`
`G-CSF
`
`GM-CSF
`
`No cytokine
`
`Cytokine
`
`3
`4
`5
`3
`4
`5
`3
`4
`5
`
`3
`4
`5
`3
`4
`5
`3
`4
`5
`
`40.9
`19.6
`34.9
`65.8
`33.3
`56.0
`69.0
`44.5
`70.5
`
`29.4
`35.4
`60.7
`41.4
`41.5
`74.5
`51.3
`52.0
`82.7
`
`± 2.4
`± 0.4
`± 4.3
`± 3.4
`± 0.6
`± 2.8
`± 1.3
`± 4.8
`± 4.1
`
`± 1.8
`±
`1.3
`± 4.1
`±
`1.6
`1.7
`±
`± 5.1
`± 0.5
`± 1.8
`± 3.5
`
`43.6
`24.2
`32.2
`55.0
`32.7
`46.4
`63.5
`45.8
`57.8
`
`26.2
`29.1
`53.9
`35.6
`34.0
`61.5
`46.0
`42.9
`78.0
`
`1.1
`±
`1.1
`±
`± 5.5
`± 3.4
`±
`1.7
`± 2.1
`± 0.7
`± 3.5
`± 2.5
`
`± 0.6
`±
`1.1
`± 4.4
`± 2.0
`± 1.2
`± 4.4
`± 0.7
`±
`1.0
`± 6.7
`
`41.5
`15.5
`
`56.5
`27.3
`
`61.7
`40.4
`
`± 2.0
`± 0.5
`ND!)
`1.7
`±
`± 0.7
`ND”
`± 2.6
`± 4.4
`ND”
`
`26.5
`28.2
`48.6
`37.3
`33.0
`59.4
`49.6
`36.7
`75.9
`
`± 0.9
`± 1.0
`± 4.9
`± 0.7
`± 2.1
`± 2.5
`±
`1.4
`± 2.1
`±
`1.3
`
`5.9
`3.3
`7.1
`6.1
`5.5
`10.2
`6.5
`5.3
`13.9
`
`4.4
`3.6
`0.6
`5.8
`5.2
`2.1
`6.3
`5.0
`10.2
`
`± 0.5
`± 0.9
`±
`1.8
`±
`1.5
`± 0.4
`±
`1.1
`± 1.2
`±
`1.1
`±
`1.3
`
`± 0.5
`± 0.9
`± 0.1
`± 0.7
`± 0.5
`± 0.2
`± 0.4
`± 0.8
`± 0.2
`
`“ Monocytes
`h ND,
`not
`
`were
`done.
`
`se
`
`parated
`
`from
`
`three
`
`different
`
`donors.
`
`Percentage
`
`of
`
`target
`
`cell
`
`lysis
`
`is expressed
`
`as mean
`
`±
`
`SD of
`
`triplicates.
`
`Table
`
`4
`
`Inhibition
`
`251
`o f BsAb
`parental
`
`X 22-mediated
`antibody”
`
`cyto
`
`toxicity
`
`by
`
`its
`
`Inhibition
`
`(%)
`
`Experiment
`
`mAb
`
`251
`
`mAb
`
`22
`
`1
`2
`
`90
`86
`
`95
`96
`
`been
`has
`humanized
`is
`
`feasible.
`Activation
`for
`the
`cial
`preincubation
`
`humanized
`BsAb
`
`for
`
`the
`Therefore,
`as well.5
`immunotherapy
`targeting
`
`of
`construction
`CD33
`and CD64
`
`a
`
`immune
`of
`cytotoxicity
`with
`
`cytokines
`
`cells
`effector
`cells.
`of
`target
`had much
`
`by
`
`cytokines
`Monocytes
`less
`cytotoxicity
`
`cru-
`was
`without
`than
`
`IFN-’y
`
`concentration
`a The
`of
`each
`concentration
`was
`these
`experiments
`100%,
`as
`22 was
`defined
`cells
`with
`IFN--y-activated
`as
`0%.
`The
`percentage
`
`I
`
`jig/mb,
`X 22 was
`251
`of BsAb
`The
`E:T
`10
`jig/mb.
`was
`parental
`mAb
`by BsAb
`mediated
`The
`50:1.
`cytotoxicity
`by mixing
`observed
`the
`and
`cytotoxicity
`any
`antibody
`was
`monocytes
`without
`inhibition
`was
`calculated
`accordingly.
`
`of
`
`the
`in
`X
`
`and
`ratio
`251
`target
`defined
`
`did
`
`those
`
`with
`
`preincubation.
`
`Although
`
`incubation
`in vivo
`study,
`incu-
`of
`
`application
`its
`FcyRI,
`of
`expression
`highest
`the
`yielded
`studied
`extensively.
`In this
`not
`been
`has
`purpose
`for
`this
`IFN-(cid:1)y,
`G-CSF,
`and GM-CSF
`effect
`of
`the
`we
`compared
`of
`Fc(cid:1)yRI
`and
`cytotoxie
`activity
`expression
`the
`bation
`on
`incubation,
`all
`three
`cytokines
`signifi-
`After
`24-h
`monocytes.
`cantly
`enhanced
`the
`cytotoxicity
`of monocytes
`in the
`presence
`of BsAb
`251
`The
`cytotoxicity
`of
`target
`cells
`by mono-
`22.
`
`x
`
`cells
`mAbs
`patients
`effector
`several
`
`leukemia
`toward
`have
`alone
`with AML
`mechanisms
`disadvantages:
`
`a BsAb.
`via
`cells
`for
`their
`studied
`been
`malignancies
`other
`and
`(20,
`21). Unconjugated
`one
`is
`that
`the
`binding
`
`Unconjugated
`antitumor
`by ADCC
`mAbs
`of
`
`and
`G-CSF
`with
`incubated
`cytes
`IFN--y.
`with
`cells
`incubated
`these
`on monocytes
`significantly
`increased
`h, but
`on monocytes
`incubated
`24
`
`not
`
`GM-CSF
`The
`
`that
`
`comparable
`is
`expression
`of Fe-yRI
`incubated
`with
`IFN--y
`with
`G-CSF
`or GM-CSF
`
`of
`was
`for
`
`for
`
`24
`
`h.
`
`However,
`
`the
`
`expression
`
`of
`
`Fe-yRI
`
`was
`
`increased
`
`significantly
`
`has
`
`been
`
`G-CSF
`
`on monocytes
`
`incubated
`
`with
`
`previously
`or GM-CSF
`
`shown
`
`in
`
`vitro
`
`that
`could
`
`incubation
`increase
`
`48
`
`for
`G-CSF
`of monocytes
`their
`cytotoxic
`
`It
`
`h.
`with
`ac-
`
`mouse
`in
`effects
`other
`and
`from
`suffer
`to Fe
`a mAb
`by
`circulating
`competed
`be
`binding
`of circulating
`immu-
`effector
`cells
`can
`block
`the
`antibody.
`This
`problem
`can
`because
`mAb
`binds
`with
`22
`binding
`site
`domain
`of
`of Fe
`can
`bind
`and
`activate
`X 22
`high
`concentration
`
`of
`
`the
`
`of
`
`may
`cells
`effector
`on
`receptors
`nonspecific
`This
`immunoglobulin.
`on
`immune
`Fc-1’RI
`to
`noglobulin
`by unconjugated
`triggered
`ADCC
`be
`overcome
`by BsAb
`251
`X
`high
`affinity
`to Fe-yRI
`outside
`immunoglobulins
`(22).
`BsAb
`Fc’yRI
`without
`interference
`
`the
`
`22
`the
`251
`
`circulating
`mouse
`antimouse
`the
`mAb.
`
`immunoglobulin.
`they
`is
`that
`mAbs
`thereby
`antibody,
`The
`development
`
`other
`The
`induce
`may
`abolishing
`of
`recombinant
`
`disadvantage
`major
`human
`the
`formation
`of
`the
`therapeutic
`effect
`humanized
`versions
`
`of
`
`of
`
`of
`
`mouse
`
`mAbs
`
`can
`
`overcome
`
`this
`
`problem.
`
`A
`
`humanized
`
`9),
`be
`
`7,
`(5,
`could
`been
`
`tivity
`Fc-yRI
`has
`also
`used
`for
`vitro
`in
`These
`observations
`immunotherapeutie
`have
`been
`CSF
`
`that
`
`showed
`data
`recent
`and
`infusion
`after
`increased
`IFN--y-activated
`reported
`of
`clonogenie
`purging
`important
`for
`clinical
`are
`approach,
`because
`both
`widely
`used
`after
`chemotherapy
`
`the
`that
`of G-CSF
`in
`monocytes
`leukemic
`application
`G-CSF
`
`expression
`
`vivo
`
`cells
`
`and
`and/or
`
`of
`It
`(9).
`be
`can
`(23).
`of our
`GM-
`bone
`
`version
`
`of
`
`anti-CD33
`
`mAb
`
`has
`
`been
`
`reported
`
`(14),
`
`and
`
`mAb
`
`22
`
`5 R. Graziano,
`
`personal
`
`communication.
`
`
`
`Downloaded from on August 31, 2015. © 1995 American Association for Cancerclincancerres.aacrjournals.org
`
`
`Research.
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1014 Page 5 of 8
`
`

`
`immunotherapy
`Although
`
`minimal
`eliminate
`to
`the majority
`of AML
`patients
`
`residual
`will
`
`leukemia
`enter
`a
`
`cells.
`clinical
`
`number
`of
`a significant
`chemotherapy,
`standard
`after
`remission
`of BsAb
`administration
`The
`eventually.
`relapse
`will
`patients
`of G-CSF
`or GM-CSF,
`will
`combination
`X 22, with
`the
`251
`of
`hematopoiesis
`and,
`at
`the
`same
`time,
`enhance
`the
`recovery
`immune
`effector
`cells
`to
`eliminate
`the
`mobilize
`and
`direct
`BsAb
`251
`residual
`leukemic
`cells.
`X 22
`also
`may
`play
`a role
`in
`bone
`marrow
`the
`setting
`of
`allogeneic
`transplantation
`by
`aug-
`it
`menting
`graft-versus-leukemia
`effect.
`Finally,
`can
`be
`applied
`bone
`marrow
`purging
`with
`IFN--y-activated
`vivo
`monocytes
`remove
`contaminating
`leukemic
`cells.
`It
`is very
`likely
`that
`strategies
`will
`work
`best
`in
`those
`situations
`in
`which
`tumor
`numbers
`are
`low and
`immune
`effector
`cells
`are
`abundant.
`
`the
`in ex
`to
`these
`cell
`
`REFERENCES
`
`Fanger,
`1.
`antibodies.
`
`M. W., Morganelli,
`Crit.
`Rev.
`Immunob.,
`
`and
`P. M.,
`101-124,
`12:
`
`Guyer,
`1992.
`
`P. M.
`
`Bispecific
`
`R.
`
`I. A., Geerars,
`J., Bast,
`B.
`malignancy
`in end
`stage
`
`J.,
`using
`leukemia
`
`A.
`and
`
`J.,
`
`de Lau, W. B., Clark,
`de Gast,
`B. C. Killing
`CD3
`X CD19
`bispecific
`and
`lymphoma.
`Blood,
`
`de
`
`van
`M. R.,
`of
`autobogous
`monocbonal
`84:
`556-563,
`
`1324 AML
`
`Cell Lysis
`
`in the Presence
`
`of BsAb
`
`251
`
`X
`
`22
`
`A. NB4
`
`cells
`
`B. HL-60
`
`cells
`
`100
`
`80(cid:1)
`
`60(cid:1)
`
`40-
`
`20-
`
`100-
`
`(cid:1)80-
`
`t15
`
`Vrj
`
`‘S
`
`0E
`
`(cid:1)20-
`
`I
`12:1
`E:T
`
`I
`25:1
`cell
`
`I
`50:1
`ratio
`
`I
`12:1
`E:T
`
`I
`25:1
`cell
`
`I
`50:1
`ratio
`
`4
`
`the
`of
`fragment
`single
`
`a
`
`22
`
`the
`similar
`
`SE.
`
`251 X 22,
`by BsAb
`mediated
`cytotoxicity
`Comparison
`Fig.
`and controls. Monocytes
`were
`of mAb
`251,
`F(ab’)2
`mAb
`251,
`donor
`and
`incubated
`with
`IFN-y.
`In
`controls
`separated
`from
`monocytes
`were
`mixed
`with
`target
`cells
`without
`(A),
`IFN--y-activated
`X 22 (#{149}),mAb
`concentrations
`of BsAb
`251
`any antibody.
`The
`251
`(Ky),
`and F(ab’)2
`fragment
`of mAb
`(0) were
`1 jig/mb.
`The
`presence
`of
`251
`mAb
`251
`and
`F(ab’)2
`fragment
`of mAb
`251
`did
`not
`increase
`the
`cytotoxicity
`compared
`with
`that
`of
`controls.
`The
`addition
`of BsAb
`251
`increased
`cytotoxicity
`significantly.
`One
`of
`three
`such
`X
`experiments
`with
`results
`is
`shown.
`Bars,
`
`to
`
`stimulate
`
`the
`
`recovery
`
`of myeboid
`
`hand,
`
`transplantation
`marrow
`hematopoiesis.
`is
`antigen
`the CD33
`of
`expression
`the
`other
`On
`the
`pro-
`and myeloid
`monocytes,
`leukemia,
`restricted
`to myeloid
`using
`trial
`in AML
`patients
`clinical
`genitor
`cells.
`A recent
`mAb
`version
`of
`anti-CD33
`humanized
`‘311-conjugated
`to
`a
`and
`cells
`efficient
`cytoreduc-
`to target
`showed
`rapid
`localization
`systemic
`toxicity
`(14).
`Anti-
`with
`little
`tion
`of
`leukemie
`blasts
`linked
`to nicin
`toxin
`on in combination
`mAb
`covalently
`CD33
`and
`chemotherapy
`has
`been
`used
`with
`rabbit
`complement
`of
`these
`findings
`purge
`bone
`marrow
`vivo
`ex
`(24-26).
`All
`be
`an excellent
`molecule
`for
`targeted
`may
`suggest
`that
`the CD33
`the
`expression
`of CD33
`on the mono-
`note,
`immunotherapy.
`Of
`with
`lower
`surface
`density
`than
`on
`cyte
`effeetor
`(albeit
`cells
`cell
`AML
`not
`cells)
`experiments
`we
`herein
`did
`affect
`in the
`report
`In
`their
`ability
`to
`target
`CD33-positive
`leukemia
`cells.
`other
`words,
`the
`possibility
`that
`the CD33
`molecules
`on monocytes
`did
`as
`could
`act
`targets
`their
`own
`cytotoxicity
`not
`appear
`for
`ADCC
`an
`be
`obstacle.
`there
`was
`any
`mediated
`Whether
`monocytes
`against
`remains
`unknown:
`we
`intend
`monocytes
`study
`this
`possibility
`future.
`Although
`the maximal
`killing
`in the
`of
`target
`cells
`required
`higher
`E:T
`ratios,
`significant
`tumor
`cell
`E:T
`lysis
`was
`observed
`at
`lower
`ratios.
`Because
`the
`cytotoxicity
`time
`in an ADCC
`assay
`is measured
`by
`choosing
`a window
`of
`in the
`ongoing
`cytotoxie
`process,
`it probably