`
`1. 425-434,
`
`April
`
`1995
`
`Clinical
`
`Cancer
`
`Research
`
`425
`
`Immunoconjugate
`An
`22 Can
`Specifically
`and
`Neutrophil-mediated
`of
`the Lung
`Cells1
`
`and Monoclonal
`of Lys3-Bombesin
`Induce
`FcyRI
`(CD64)-dependent
`Lysis
`of Small
`Cell
`Carcinoma
`
`Antibody
`Monocyte-
`
`Chen,
`
`Jian
`Michael
`Division
`Pittsburgh
`Pennsylvania
`Pharmaceutical
`Pennsylvania
`Dartmouth
`[M. W. F.]
`
`Zhou,
`Jie-Hua
`Mokotoff,
`Michael
`D. Ball2
`and
`Edward
`Fanger,
`W.
`Marrow
`Transplantation,
`of Hematology/Bone
`and Pittsburgh
`Cancer
`Institute,
`Medical
`Center
`J-H. Z., E. D. B.]; Department
`15213
`[J. C.,
`Sciences,
`University
`of Pittsburgh,
`Pittsburgh,
`15213
`[M. M.];
`and
`Department
`of Microbiology,
`Medical
`School,
`Lebanon,
`New Hampshire
`03756
`
`of
`
`University
`Pittsburgh,
`of
`
`INTRODUCTION
`
`The
`advances
`mated
`States
`
`SCCL3
`of
`
`recent
`despite
`poor
`remains
`for
`prognosis
`esti-
`An
`cancers.
`other
`certain
`in the management
`the United
`in
`diagnosed
`were
`SCCL
`42,500
`cases
`of
`cancers
`(1,
`2).
`25% of
`all
`lung
`for
`in
`1993,
`accounting
`to chemother-
`patients
`respond
`the majority
`of SCCL
`Although
`survival
`response
`is of
`short
`duration
`(3, 4). Median
`apy,
`the
`and
`with
`limited-stage
`disease
`is
`14-18
`months,
`patients
`15-25%
`with
`extensive
`disease
`is 9-1
`1 months.
`Only
`patients
`(1-4).
`of
`limited-stage
`patients
`survive
`for more
`than
`2 years
`used
`Many
`combinations
`of
`chemotherapeutic
`agents
`have
`been
`clinically
`without
`much
`improvement
`in the
`long-term
`survival
`rate.
`Recent
`efforts
`have
`been
`focused
`on
`the
`development
`other
`therapeutic
`strategies
`for
`the
`treatment
`of SCCL,
`including
`immunological
`and
`hormonal
`therapy
`(5,
`and
`intensive
`che-
`6)
`motherapy
`autologous
`bone marrow
`or peripheral
`stem ceil
`support
`
`for
`for
`
`of
`
`with
`(7-9).
`
`isolated
`initially
`was
`which
`peptide
`acid
`a 14-amino
`is
`BN
`(10).
`The mamma-
`bombina
`Bombina
`frog
`skin
`of
`the
`from the
`GRP,
`contains
`a COOH-terminal
`hep-
`han
`analogue
`of BN,
`tapeptide
`sequence
`identical
`to that
`of BN (1 1). The majority
`of
`human
`SCCL
`cell
`lines
`produce
`GRP
`and
`express
`a single
`class
`of high-affinity
`receptors
`for BN/GRP
`(12-16).
`It has
`also
`been
`demonstrated
`that GRP
`functions
`as
`autocrine
`growth
`factor
`an
`for
`human
`SCCL
`cells.
`Blockage
`of
`autocrine
`growth
`stim-
`this
`ulatory
`activity
`of GRP
`in SCCL
`has
`been
`the
`focus
`of
`several
`studies.
`A mAb
`against
`GRP
`and
`a number
`of BN/GRP
`receptor
`antagonists
`have
`been
`shown
`to
`inhibit
`the
`growth
`of
`SCCL
`cells
`both
`Since
`BN/GRP
`and
`in
`vitro
`in
`vivo
`(5,
`17-19).
`receptors
`have
`limited
`distribution
`in the
`body,
`they
`serve
`can
`targets
`for
`specific
`immune
`reactions.
`A novel
`approach
`immunotherapy
`targeting
`the BN/GRP
`receptors
`expressed
`SCCL
`cell
`surface
`has
`been
`developed
`in our
`laboratory.
`have made
`an immunoconjugate
`between
`a BN-like
`peptide
`a mAb
`is
`expressed
`neutrophils.
`
`against
`
`human
`
`high-affinity
`
`FcyR
`
`(Fc-yRI,
`
`CD64),
`
`which
`
`on
`We
`
`surface
`the
`hypothesized
`
`of
`
`human
`that
`this
`
`monocytes
`immunoconjugate
`
`and
`
`activated
`should
`
`as
`of
`on
`We
`and
`
`for
`It
`is
`a
`
`accounts
`prognosis.
`peptide,
`of bom-
`pep-
`
`as
`
`of
`of
`
`ABSTRACT
`(SCCL)
`lung
`the
`of
`carcinoma
`cell
`Small
`a very
`has
`and
`lung
`of all
`poor
`cancers
`25%
`gastrin-releasing
`produce
`cells
`SCCL
`that
`known
`to that
`actions
`biological
`similar
`has
`which
`peptide
`gastrin-releasing
`of
`counterpart
`an
`amphibian
`besin,
`surface
`bombesin/gastrin-
`cell
`affinity
`high
`and
`express
`tide,
`receptors
`These
`receptors.
`releasing
`peptide
`can
`serve
`Cell
`surface
`receptors
`immunotherapy.
`for
`specific
`targets
`a family
`of molecules
`are
`of
`IgG (FcyR)
`for
`Fc portion
`the
`reactions,
`including
`immune
`a variety
`of
`that
`can mediate
`that
`immuno-
`tumor
`cell
`cytotoxicity.
`We
`hypothesized
`an
`directed
`the
`high-
`conjugate
`of
`bombesin
`and
`a mAb
`to
`able
`to
`specific
`should
`be
`trigger
`affinity
`FcyRI
`(mAb
`22)
`cytotoxicity
`against
`cells.
`In this
`article,
`report
`the
`SCCL
`we
`of
`this
`immunoconjugate
`and
`demonstrate
`construction
`its
`capacity
`redirect
`immune
`effector
`cells
`toward
`SCCL
`of
`cells
`and
`lysis
`these
`target
`cells.
`The
`immunoconju-
`lines
`gate
`stained
`the majority
`of cells
`from four
`SCCL
`cell
`neu-
`and
`reacted
`with
`on activated
`monocytes
`and
`Fc(cid:1)yRI
`neutrophils
`trophils.
`After
`preincubating
`monocytes
`and
`with
`recombinant
`‘y interferon
`to enhance
`the
`expression
`cell
`on the
`surface,
`we demonstrated
`that
`60-98%
`FcyRI
`SCCL
`cells
`could
`be
`lysed
`in the presence
`of
`the
`immuno-
`conjugate
`in a chromium
`release
`assay.
`Tumor
`cell
`lysis was
`range
`over
`a wide
`of
`immunoconjugate
`concentra-
`observed
`be
`on the
`tions,
`dependent
`ratio
`of E:T
`cells,
`and
`could
`the
`of
`by
`the
`addition
`either
`parental
`molecule
`of
`blocked
`immunoconjugate.
`Bispecific
`molecules
`redirecting
`immune
`efTector
`cells
`target
`SCCL
`cells
`may
`have
`clinical
`appli-
`to
`of SCCL.
`cation
`the
`therapy
`
`to
`elicit
`
`was
`
`in
`
`Received
`C This work
`
`8/26/94;
`was
`
`accepted
`supported
`
`12/12/94.
`by Grant
`
`CA37868
`
`from the National
`
`Cancer
`
`NIH,
`Institute,
`2 To whom
`
`Bethesda,
`requests
`
`for
`
`MD.
`reprints
`
`should
`
`be
`
`addressed,
`
`at Division
`
`of
`
`Hematology/Bone
`pital,
`200
`Lothrop
`
`Marrow
`Street,
`
`Transplantation,
`Pittsburgh,
`
`PA
`
`Montefiore
`15213.
`
`University
`
`Hos-
`
`are: SCCL,
`used
`abbreviations
`gastrin-releasing
`GRP,
`bombesin;
`cell-mediated
`antibody-dependent
`Sulfo-SMCC,
`S-acetylthioacetate;
`cydlohexane-1-carboxylate;
`free
`sulfhydryl
`group;
`fluorescence
`intensity;
`PBA,
`phosphate-buffered
`
`lung;
`the
`of
`carcinoma
`cell
`small
`receptor;
`Fc
`Fc-yR,
`peptide;
`-y
`N-sue-
`SATA,
`cytotoxicity;
`4-(N-male-
`sulfosuccinimidyl
`R-HPLC,
`reversed
`phase
`human
`recombinant
`IFN--y;
`rIFN--y,
`granulocyte-colony-stimu-
`G-CSF,
`saline-bovine
`serum
`albumin-
`
`3 The
`BN,
`ADCC,
`cinimidyl
`imidomethyl)
`HPLC;
`SH,
`MFI, mean
`lating
`factor;
`sodium
`aside.
`
`
`
`Downloaded from on August 31, 2015. © 1995 American Association for Cancerclincancerres.aacrjournals.org
`
`
`Research.
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1016 Page 1 of 11
`
`
`
`426
`
`Immunoconjugate
`
`of Lys3-BN
`
`and mAb
`
`22 Can
`
`Induce
`
`Lysis
`
`of SCCL
`
`Lys3-BN
`
`+
`
`Ac(cid:1)S(cid:1)CH2COO_NO
`
`NH2
`
`SATA
`
`Lys3-BN
`
`(cid:1)H
`
`-C0-CH2-S-Ac
`
`IN H 2 0 H
`
`Lys3-BN
`
`NH-C0-CH2-
`
`SH
`
`0
`
`.1/
`0
`
`2
`
`mAb22
`
`NaO3S
`
`0
`
`/1
`
`tN
`
`-
`
`0 C 0 (cid:1)
`
`C
`
`-
`
`0
`
`Sulfo-SIICC
`
`0
`\\
`
`Ii
`0
`
`,(cid:1)
`
`mAb22
`
`HN-C0
`
`0
`
`1/
`0
`
`mAb22
`
`HN-C0(cid:1)CH,-N(cid:1)
`
`0
`\\
`
`7T(cid:1)(cid:1)’Ns
`
`-CH2CONH
`
`Lys3-BN
`
`0
`
`stAb22-Lys-BN
`
`iaaunocon)ugate
`
`Fig.
`
`I
`
`Schema
`
`of
`
`the
`
`chemical
`
`conjugation
`
`between
`
`Lys3-BN
`
`and mAb
`
`22
`
`or
`
`its F(ab’),
`
`fragments.
`
`be
`cells
`
`able
`and
`
`to
`
`these
`redirect
`elicit
`specific
`
`immune
`ADCC
`
`effector
`against
`these
`
`cells
`cells.
`
`toward
`
`SCCL
`
`room
`
`the
`
`at
`
`(pH
`
`was
`
`(GIBCO-BRL)
`din
`CO2. Another
`Waymouth’s
`FCS.
`with
`10%
`Antibodies
`F(ab’)2
`
`MATERIALS
`Cell
`Lines.
`SHP-77,
`were
`BRL,
`Grand
`L-glutamine,
`
`AND METHODS
`SCCL
`cell
`lines,
`maintained
`in RPMI
`Island,
`NY)
`supplemented
`and
`100
`units/ml
`penicillin,
`at 37#{176}Cin a humidified
`cell
`line, DMS273
`SCCL
`(20),
`MB
`752/1
`medium
`(GIBCO-BRL)
`
`and
`NCI-H345,
`NCI-H69,
`(GIBCO-
`medium
`1640
`10% FCS,
`2 mr(cid:1)t
`with
`p.g/ml
`streptomy-
`100
`atmosphere
`with
`was
`maintained
`supplemented
`
`5%
`in
`
`22
`
`21),
`mAb
`were
`an
`SCCL-1,
`was
`produced
`
`and
`fragments
`
`Reagents.
`of mAb
`
`Anti-Fc-yRI
`(F(ab’)2),
`
`22;
`
`(mAb
`FITC-labeled
`
`and
`
`22
`
`from
`
`obtained
`IgG2a
`mAb
`reactive
`in this
`laboratory.
`
`Medarex,
`
`Inc.
`
`(Annandale,
`
`receptor
`transferrin
`with
`Lys3-BN,
`a BN analogue
`
`Ref.
`
`NJ).
`
`(22),
`with
`
`similar
`
`binding
`
`affinity
`
`to
`
`the
`
`BN/GRP
`
`receptor
`
`(1 1,
`
`13),
`
`and
`
`hydroxylamine
`
`were
`
`purchased
`
`from
`
`Sigma
`
`Chemical
`
`Company
`
`Louis,
`
`Conjugation
`MO).
`obtained
`from
`
`were
`
`chemicals,
`Pierce
`Chemical
`
`SATA
`Co.
`
`Sulfo-
`and
`(Rockford,
`
`(St.
`SMCC,
`IL).
`
`Protein
`M sodium
`0.1
`and
`EDTA
`ylformamide.
`
`Conjugation.
`phosphate
`SATA
`The
`SATA
`
`the
`
`Lys3-BN
`7.4)
`buffer
`(pH
`freshly
`dissolved
`was
`was mixed
`with
`L-BN
`
`was
`
`freshly
`
`dissolved
`
`in
`
`m(cid:1)i
`2.5
`containing
`dimeth-
`in
`100%
`in a final molar
`
`temperature,
`at
`reaction
`30 mm of
`10:1. After
`of
`ratio
`from
`nonreacted
`was
`separated
`conjugate
`Lys3-BN-SATA
`C18
`analytical
`SATA
`by R-HPLC
`on
`a Vydac
`Lys3-BN
`and
`R-HPLC
`eluent
`containing
`Lys3-BN-SATA
`was
`column.
`The
`adjusted
`to pH 7.0
`adding
`1 M sodium
`by
`The
`free
`sulthydryl
`group
`was
`generated
`hydroxylamine
`4#{176}Cfor
`2 h. A second
`
`phosphate
`by
`deacetylation
`R-HPLC
`
`8.0).
`with
`per-
`
`The
`
`Lys3-BN-SH.
`separate
`to
`formed
`and
`neutralized
`collected
`was
`BN-SH
`could
`be
`groups
`sulthydryl
`free
`the
`same
`At
`Ellman’s
`reagent.
`reacted
`22 was
`ments
`of mAb
`antibody.
`maleimide-activated
`Sulfo-SMCC
`arated
`from unreacted
`(Amicon,
`Centricon
`30 apparatus
`jugation
`between
`Lys3-BN-SH
`carried
`out
`by mixing
`an
`equal
`ature
`overnight.
`The
`unreacted
`were
`removed
`by
`centrifugation
`ratus.
`The
`concentration
`of
`the
`
`fraction
`containing
`to pH 7.0. The
`presence
`determined
`via
`reaction
`time,
`mAb
`or F(ab’)2
`22
`with
`Sulfo-SMCC
`to produce
`The
`activated
`antibody
`was
`by centrifugation
`through
`con-
`Beverly,
`MA).
`final
`The
`was
`and
`the
`activated
`antibody
`at
`temper-
`molar
`amount
`room
`other
`Lys3-BN
`and
`by-products
`through
`a Centricon
`30
`appa-
`immunoconjugate
`was
`quantified
`
`Lys3-
`
`of
`with
`frag-
`
`sep-
`
`a
`
`a
`
`using
`mond,
`scheme
`
`with
`(PBA
`
`DC
`a Bio-Rad
`its
`and
`CA)
`peptide
`the
`for
`Immunofluorescence.
`ice-cold
`PBS
`containing
`solution)
`and
`incubated
`
`assay
`protein
`by
`was
`purity
`shown
`conjugation
`SCCL
`cells
`0.1% BSA and
`with
`different
`
`(Bio-Rad
`checked
`is
`
`Laboratories,
`SDS-PAGE.
`in Fig.
`1.
`were
`washed
`0.1% sodium
`amounts
`
`Rich-
`The
`
`twice
`azide
`of
`the
`
`
`
`Downloaded from on August 31, 2015. © 1995 American Association for Cancerclincancerres.aacrjournals.org
`
`
`Research.
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1016 Page 2 of 11
`
`(cid:1)
`(cid:1)
`(cid:1)
`
`
`50
`
`100
`
`the
`by
`
`a
`
`to
`
`in
`
`and
`
`22
`were
`San
`cell
`
`for
`
`p.g/ml
`of
`presence
`1 h in the
`at 4#{176}Cfor
`immunoconjugate
`added
`immunoconjugate
`final
`concentration
`of
`The
`human
`IgG.
`three
`jig/mI
`per
`106 cells.
`After
`washing
`and
`was
`5, 25,
`5 X
`cells
`incu-
`solution,
`the
`were
`resuspended
`and
`PBA
`times
`with
`F(ab’)2
`FITC-labeled
`goat
`anti-mouse
`immunoglob-
`bated
`with
`Francisco,
`Lab.,
`South
`San
`CA)
`for
`30 mm at 4#{176}C.
`ulin
`(Caltag
`fixed
`cells
`were
`1% paraformaldehyde.
`the
`After
`washing,
`neutrophils,
`before
`after
`IFN-y
`stimulation,
`Monocytes
`and
`were
`stained
`directly
`with
`FITC-Iabeled
`mAb
`to evaluate
`expression
`of
`FcyRI.
`All
`of
`the
`samples
`analyzed
`CA).
`FACScan
`flow
`cytometry
`(Becton
`Dickinson,
`Jose,
`deter-
`per
`The
`number
`of
`antibody
`sites
`bound
`was
`with
`containing
`by
`using
`a standard
`mixture
`beads
`mined
`bead
`six
`different
`concentrations
`of FITC
`labeling
`(23).
`The
`standard
`from
`beads
`were
`purchased
`Flow
`Cytometry
`Standards
`Corpo-
`Rico;
`ration
`(San
`Juan,
`Puerto
`Quantum
`24 and
`25).
`The median
`the
`of
`fluorescent
`intensities
`six
`bead
`peaks
`were
`used
`to set
`determination
`standard
`curve
`for
`of
`the
`amount
`of
`FITC/cell.
`by
`calculated
`the
`Antibody/cell
`was
`dividing
`the
`FITC/cell
`by
`the
`ratio
`for
`flourescein:protein
`standard
`bead mixture.
`Appro-
`negative
`priate
`isotype-matched
`controls
`were
`performed
`binding
`of
`subtract
`nonspecific
`antibody.
`to
`Binding
`Stability
`of Lys3-BN
`x 22 Immunoconjugate
`Cells.
`It has
`SCCL
`are
`receptors
`been
`reported
`that
`BN/GRP
`deter-
`(24).
`To
`internalized
`upon
`binding
`BN or GRP
`rapidly
`cells
`SCCL
`the
`stability
`of
`immunoconjugate
`binding,
`mine
`30 mm and
`incubated
`with
`20 p.g/ml
`were
`immunoconjugate
`The
`immu-
`then washed
`to remove
`unbound
`immunoconjugate.
`noconjugate-bound
`cells were
`incubated
`at 37#{176}Cin fresh
`media
`for
`various
`times
`before
`the
`addition
`of FITC-Iabeled
`antimouse
`antibody
`at 4#{176}C.The
`binding
`of
`the
`immunoconjugate
`to SCCL
`the
`cells
`at
`initiation
`of
`incubation
`at 37#{176}Cwas
`defined
`as 100%.
`Isolation
`of
`EfTector
`from
`Peripheral
`Blood.
`Cells
`Monocytes
`separated
`from Leuko-Packs
`obtained
`from the
`Pittsburgh
`Blood
`Bank.
`After
`Ficoll-Hypaque
`gradient
`centrifugation,
`mononuclear
`cells
`were
`washed
`twice
`with
`HBSS
`(GIBCO-BRL)
`containing
`1 mr(cid:1)t EDTA
`and
`then
`cultured
`
`were
`Central
`
`10%
`
`FCS
`The
`anti-CD14,
`(Becton
`to
`
`2 h
`for
`adherent
`anti-
`Dickin-
`determine
`donors.
`
`containing
`medium
`1640
`RPMI
`with
`a flask
`in
`were
`removed.
`cells
`nonadherent
`37#{176}C.The
`at
`stained
`with
`were
`detached,
`collected,
`cells
`and
`anti-CD56
`anti-CD3,
`anti-CD13,
`CD45,
`flow
`cytometry
`and
`analyzed
`by
`FACScan
`son),
`A
`isolated
`from normal
`purity.
`The
`neutrophils
`were
`the
`The
`20-ml
`blood
`sample
`was
`centrifuged
`Ficoll-Hypaque.
`on
`was
`1640
`cell
`pellet
`suspended
`in
`an
`equal
`volume
`of RPMI
`Four
`medium.
`hundred
`p.1 of 5% dextran
`were
`added
`to each ml
`sample.
`of blood
`After
`sedimentation
`at 1 X g
`for
`
`1 h at 4#{176}C,the
`neutrophil-rich
`supernatant
`was
`collected.
`The
`purity
`of neutro-
`phils
`thus
`isolated
`was
`usually
`higher
`95%.
`Activation
`Immune
`Effector
`Cells.
`of
`provided
`by Genentech
`(San
`Francisco,
`kindly
`of
`rIFN-’y
`used
`in this
`study
`(200
`centration
`its
`saturate
`receptors
`and
`to
`induce
`to
`shown
`in the
`expression
`of Fc(cid:1)yRI
`on
`the
`surface
`crease
`neutrophils
`(25).
`Isolated
`monocytes
`and
`and
`incubated
`with
`rIFN--y
`in RPMI
`1640 medium
`FCS
`for
`18
`h at 37#{176}Cbefore
`the ADCC
`assay.
`of Fc-yRI
`before
`and
`after
`rIFN--y
`incubation
`was
`
`than
`
`was
`con-
`been
`in-
`
`rIFN--y
`CA).
`The
`units/ml)
`has
`a maximum
`of monocytes
`neutrophils
`were
`containing
`10%
`The
`expression
`determined
`
`by
`
`Clinical
`
`Cancer
`
`Research
`
`427
`
`staining
`flow
`
`FITC-labeled
`
`mAb
`
`22
`
`and
`
`analyzed
`
`by
`
`FACScan
`
`The
`
`with
`cytometry.
`Assay.
`ADCC
`96-well
`in
`performed
`was
`assay
`Co., Woburn,
`Instrument
`(Rainin
`plates
`microtiter
`round-bottomed
`MA). The
`RPMI
`1640
`once with
`washed
`cells were
`SCCL
`target
`sodium
`[5tCr]chromate
`jiCi
`with
`100
`and
`incubated
`medium
`for
`1 h at
`37#{176}C.After
`MA)
`Boston,
`England
`Nuclear,
`(New
`resuspended
`in RPM!
`1640
`cells
`were
`several
`times,
`washing
`a
`concentration
`of
`1 X
`containing
`10% FCS
`to
`medium
`i05
`as
`neutrophils
`serving
`effector
`Activated
`monocytes
`or
`cells/ml.
`suspended
`in RPMI
`1640 medium
`in a final
`concen-
`cells were
`2 x
`cells/ml.
`Effector
`cells
`p.1) were
`added
`tration
`of
`i0(cid:1)
`row of wells
`and
`serial
`dilution
`performed
`with
`to the
`first
`an equal
`volume
`of RPMI
`1640 medium.
`100
`p.1 of
`target
`cells
`were
`added
`in the wells
`to yield
`ratio
`of 100:1,
`
`(100
`was
`Then,
`a final
`E:T
`
`assay,
`standard
`a
`In
`12:1.
`and
`25:1,
`50:1,
`concentration
`a final
`to yield
`added
`gate were
`in each
`assay
`was
`included
`mAb
`SCCL-1
`the monocytes
`activity
`of
`the
`to measure
`100:1
`controls
`with
`E:T
`ratio
`of
`eral
`other
`each
`assay,
`including
`incubation
`rated
`in
`target
`immunoconjugate
`alone,
`incubation
`of
`target
`antibody,
`without
`any
`and
`incubation
`of
`with
`unconjugated
`mAb
`In each
`assay,
`22.
`Lys3-BN
`and
`unconjugated
`mAb
`along
`conjugate
`were
`incubated
`along
`with
`the
`target
`to determine
`whether
`tumor
`cell
`lysis
`could
`
`5
`
`as
`and
`
`immunocongu-
`jig
`The
`of 25
`jig/mI.
`a positive
`control
`neutrophils.
`5ev-
`were
`also
`incorpo-
`of
`target
`cells
`with
`and
`effector
`cells
`and
`effector
`cells
`a 10-fold
`excess
`of
`with
`the
`immuno-
`and
`effector
`cells
`be
`blocked
`by
`
`22
`
`the
`
`of
`of
`
`either
`ence
`rected
`1 mg/ml
`4 h or
`was
`lysis
`target
`
`To
`
`of
`
`cell
`
`pres-
`the
`whether
`determine
`molecule.
`parental
`the
`redi-
`the
`with
`could
`interfere
`human
`immunoglobulin
`of
`concentration
`IgG at a final
`tumor
`killing,
`human
`cell
`After
`incubation
`at 37#{176}Cfor
`was
`in some
`assays.
`added
`the
`h,
`the microplates
`were
`centrifuged
`and
`supernatant
`18
`collected
`for
`the
`determination
`of 51Cr
`release.
`Maximum
`the
`addition
`100
`p.1 5% NP4O
`was
`achieved
`by
`to
`100
`cells.
`The
`percentage
`of
`lysis
`was
`calculated
`as:
`
`p.1
`
`(experimental
`
`cpm
`
`-
`
`spontaneous
`
`release
`
`mean
`
`cpm)
`
`x
`
`100/(maximum
`
`release mean
`
`cpm
`
`-
`
`spontaneous
`
`mean
`
`cpm)
`
`from the
`of 5tCr
`release
`Spontaneous
`release
`in a 4-h
`assay
`of maximum
`10%
`the
`dose-response
`assays,
`assay.
`For
`ratio
`and
`added.
`The
`E:T
`diluted
`amount
`immunoconjugate
`the
`Since
`100:1.
`25
`p.g/ml,
`we
`defined
`the
`was
`assay
`dard
`that
`amount
`of
`immunoconjugate
`lysis with
`cell
`cell
`lysis
`achieved
`with
`diluted
`ity. The
`tumor
`accordingly.
`was
`calculated
`Analysis.
`Statistical
`and
`from triplicates
`level
`was
`determined
`
`of
`
`than
`less
`was
`cells
`target
`20% in an
`than
`less
`and
`was
`immunoconjugate
`was
`in
`those
`assays
`added
`in a stan-
`percentage
`of
`tumor
`as
`100%
`activ-
`immunoconjugate
`
`Each
`reported
`by
`
`experimental
`as
`the mean
`paired
`Student’s
`
`result
`± SD.
`test
`
`ob-
`was
`Signif-
`when
`
`t
`
`18-h
`
`serially
`
`tamed
`icance
`applicable.
`
`RESULTS
`Binding
`Cells.
`react
`
`SCCL
`did
`not
`
`Lys3-BN
`the
`of
`Unconjugated
`with
`SCCL
`
`cells.
`
`x
`
`mAb
`After
`
`Immunoconjugate
`22
`and
`its F(ab’)2
`22
`conjugation
`with
`
`fragments
`Lys3-BN,
`
`to
`
`
`
`Downloaded from on August 31, 2015. © 1995 American Association for Cancerclincancerres.aacrjournals.org
`
`
`Research.
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1016 Page 3 of 11
`
`
`
`428
`
`lmmunoconjugate
`
`of Lys3-BN
`
`and mAb
`
`22 Can
`
`Induce
`
`Lysis
`
`of
`
`SCCL
`
`DMS273
`
`cells
`
`DMS273
`
`cells
`
`300
`
`200
`
`100
`
`0
`
`10
`
`20
`
`30
`
`40
`
`50
`
`0
`
`10
`
`20
`
`30
`
`40
`
`50
`
`H345
`
`cells
`
`H345
`
`cells
`
`300
`
`MFI
`mAb
`been
`H69
`receptor
`tometric
`
`imately
`SHP-77
`pared
`
`100
`
`(cid:1).80
`
`(cid:1).60
`
`40
`
`20
`
`0
`
`100(cid:1)(cid:1).
`
`80
`
`the
`than
`and
`22
`estimated
`cell
`lines
`numbers
`analysis,
`
`prepared
`one
`Lys3-BN.
`The
`and
`to be
`1500
`We
`(16,
`24).
`on DMS273
`it
`appears
`
`that
`
`fragments
`F(ab’),
`the
`between
`receptors
`of BN/GRP
`number
`for H345
`respectively,
`1000,
`the
`BN/GRP
`not
`quantified
`have
`By
`flow
`SHP-77
`cells.
`and
`DMS273
`cells
`express
`approx-
`
`of
`has
`and
`
`cy-
`
`the
`
`same
`
`number
`
`of
`
`BN/GRP
`
`slightly
`
`fewer
`
`BN/GRP
`
`receptors,
`
`receptors
`
`while
`
`the
`
`when
`
`com-
`
`cells
`to H345
`The
`binding
`peripheral
`tested.
`
`have
`cells.
`Lys3-BN
`the
`of
`and
`lymphocytes
`The
`immunoconjugates
`
`also
`
`normal
`was
`
`X 22
`to two
`did
`
`immunoconjugate
`leukemia
`not
`bind
`
`to
`lines
`cell
`to normal
`
`60
`
`40
`
`20
`
`0
`
`:
`
`I.’.’.
`1 0
`
`20
`
`3 0
`
`40
`
`(cid:1) 5’O
`
`D
`
`10
`
`(cid:1) 20
`
`(cid:1) (cid:1) (cid:1) 5(cid:1)0
`
`CC.
`
`0(cid:1)
`200
`100
`
`H69
`
`cells
`
`peripheral
`
`lymphocytes.
`
`stained
`
`20-50%
`
`of
`
`cells
`
`The
`
`from
`
`mAb
`
`22
`
`and
`
`its
`
`F(ab’)2
`
`fragments
`
`the
`
`HL-60
`
`and
`
`NB4
`
`leukemia
`
`cell
`
`lines
`
`with
`
`very
`
`dim
`
`fluorescence,
`
`consistent
`
`with
`
`the
`
`known
`
`of
`
`Fc-yRI
`intensity
`
`these
`on
`when
`although
`
`cells.
`these
`the
`
`There
`cells
`were
`percentage
`
`change
`with
`
`no
`was
`stained
`of
`positive
`
`in
`the
`cells
`
`H69
`
`cells
`
`100
`
`(cid:1).80
`
`>.60
`
`40
`
`(cid:1)20
`
`0
`
`0
`
`1(cid:1)0
`
`20
`
`30
`
`40
`
`50
`
`SHP-77
`
`cells
`
`100
`
`40(cid:1)
`
`(cid:1)(cid:1)(cid:1)
`
`:
`
`U.
`
`300
`
`200
`
`100
`
`0
`
`300
`
`200
`
`100
`
`0
`
`10
`
`20
`
`30
`
`40
`
`50
`
`SHP-77
`
`cells
`
`0
`
`10
`
`20
`
`30
`
`40
`
`50
`
`Concintratlon
`
`(Ng/ml)
`
`10
`20
`Concsntratlon
`
`30
`
`40
`
`50
`
`((cid:1)gIml)
`
`of
`using
`
`are
`the
`
`expression
`fluorescence
`immunoconjugate,
`increased
`slightly.
`The
`number
`calculated
`was
`an
`average
`bound
`bound
`5036,
`cells
`2399.
`The
`bound
`the
`published
`than
`Since
`there
`16).
`is
`possible
`that
`BN/GRP
`receptors
`GRP
`receptors.
`It has
`been
`internalized
`upon
`x
`of Lys3-BN
`SCCL
`lines were
`of
`immunoconjugate
`unbound
`immunoconjugate.
`fresh
`media
`for
`various
`
`receptors
`BN/GRP
`the
`that
`reported
`determine
`To
`BN/GRP.
`binding
`binding,
`immunoconjugate
`22
`a saturation
`incubated
`with
`for
`30 mm and
`then
`The
`cells were
`times
`before
`the
`
`cells
`amount
`washed
`incubated
`addition
`
`quickly
`are
`stability
`the
`from
`four
`(25
`jig/mI)
`to
`remove
`at 37#{176}Cin
`of
`FITC-
`
`cell
`SCCL
`immunoglobulin
`cells
`H345
`a
`bead
`calibration
`H69
`molecules,
`61 16
`immunoconjugate
`of
`cells
`and
`SHP-77
`9473,
`273
`cells
`bound
`DMS
`of bound
`immunoconjugates
`is greater
`number
`of BN/GRP
`receptors
`cell
`(14-
`number
`types
`of BN/GRP
`receptors
`present,
`three
`immunoconjugate
`may
`bind
`to
`low-affinity
`or
`cross-bind
`other
`family
`members
`
`molecules
`method.
`
`per
`
`per
`
`it
`
`of
`
`to
`
`cytometric
`made
`
`Flow
`2
`Fig.
`immunoconjugate
`represents
`Each
`point
`10% for
`is
`less
`than
`mouse
`IgG was
`included
`positive
`cells
`and
`the MFI
`gate
`added.
`Compared
`Lys3-BN
`and mAb
`22,
`and
`F(ab’)2
`has
`a similar
`MFI.
`
`SCCL
`four
`of
`analysis
`and mAb
`from
`Lys3-BN
`individual
`the mean
`of
`three
`for MFI.
`positivity
`and
`20%
`in
`of
`analyses.
`Both
`all
`the
`increase
`with
`the
`amount
`with
`the
`immunoconjugate
`the
`immunoconjugate
`made
`percentage
`of positivity,
`but
`
`the
`(0).
`SD
`of
`of
`
`with
`stained
`lines
`22 (#{149})
`or F(ab’),
`the
`analyses
`and
`Isotypic
`control
`the
`percentage
`of
`immunoconju-
`made
`between
`between
`Lys3-BN
`consistently
`lower
`
`three
`from
`results
`The
`antibody.
`antimouse
`labeled
`positive
`percentage
`3. The
`are
`shown
`in Fig.
`period.
`first
`the
`during
`change
`significantly
`binding
`immunoconjugate
`of
`incubation,
`the majority
`Lys3-BN
`x
`Binding
`of
`the
`Immunoconjugate
`22
`Monocytes
`and
`Neutrophils.
`We
`also
`binding
`tested
`the
`monocytes
`peripheral
`the
`Lys3-BN
`X 22
`immunoconjugate
`to
`for
`18 h.
`before
`and
`after
`incubation
`with
`units/ml
`rIFN--y
`rIFN--y
`dramatically
`increased
`the
`This
`shown
`Table
`
`of
`4-h
`
`experiments
`cells
`did
`After
`was
`
`not
`24-h
`lost.
`to
`of
`
`is
`
`in
`
`1 .
`
`200
`
`by
`
`of
`the
`of
`
`binding
`the
`detected
`The
`results
`immunoconjugates
`Fig.
`2. The
`binding
`immunoconjugate
`both
`by
`an
`positively
`population.
`from
`5-50
`increased
`< 100
`tween
`
`immunoconjugate
`the
`addition
`of FITC-labeled
`flow
`cytometric
`analysis
`to
`four
`SCCL
`cell
`was
`directly
`proportional
`This
`used
`to
`stain
`the
`cells.
`in the
`absolute
`percentage
`increase
`augmentation
`the MFI
`of
`and
`by
`amount
`of
`immunoconjugate
`As
`the
`the
`percentage
`of
`positive
`p.g/ml,
`from 50-85%,
`and
`the MFI
`generally
`to >200.
`In general,
`the
`immunoconjugate
`the whole
`antibody
`of mAb
`and
`Lys3-BN
`22
`
`an
`
`of
`
`of
`lines
`
`could
`cells
`to SCCL
`antibody.
`antimouse
`of
`two
`the
`binding
`are
`illustrated
`in
`to
`the
`amount
`of
`was manifested
`of
`cells
`stained
`the
`entire
`cell
`was
`increased
`cells
`generally
`increased
`from
`prepared
`be-
`had
`a higher
`
`be
`
`expression
`
`increase
`change
`phocytes.
`
`of
`
`Fc-yRI
`
`on
`
`human
`
`monocytes
`
`as
`
`defined
`
`of MFI
`in
`the
`The
`
`<30
`
`from
`expression
`conjugation
`
`In
`
`> 120.
`to
`on
`of FcyRI
`of
`Lys3-BN
`
`there
`contrast,
`peripheral
`human
`to mAb
`22
`
`by
`
`was
`
`did
`
`the
`
`no
`lym-
`not
`
`interfere
`
`with
`
`its
`
`binding
`
`to
`
`Fc-yRI.
`
`Neutrophils
`
`without
`
`rIFN--y
`
`not
`
`or
`rIFN-(cid:1)y
`X 22
`
`to
`bind
`18
`for
`immunocon-
`
`the
`h,
`
`FcyRl
`surface
`cell
`express
`did
`treatment
`with
`incubation
`After
`immunoconjugate.
`to the Lys3-BN
`bound
`>90%
`of
`neutrophils
`50.
`of
`MFI
`jugate
`with
`a median
`Tumor
`Redirected
`Immunoconjugate
`Lys3-BN
`x
`22
`Lysis.
`Cell
`ability
`of
`the
`Lys3-BN
`The
`X 22
`immunoconju-
`to elicit monocyte-mediated
`and
`neutrophil-mediated
`gate
`lysis
`was
`tested
`in
`of
`5tCr
`release
`cell
`series
`optimal
`concentration
`of
`the
`immunoconjugate-induced
`
`a
`
`assays.
`
`tumor
`The
`cyto-
`
`
`
`Downloaded from on August 31, 2015. © 1995 American Association for Cancerclincancerres.aacrjournals.org
`
`
`Research.
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1016 Page 4 of 11
`
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`(cid:1)
`
`
`Clinical
`
`Cancer
`
`Research
`
`429
`
`120
`
`-
`
`1 00
`
`(cid:1) (cid:1)
`
`DMS273
`
`80
`
`60
`
`40-
`
`2:.
`
`H345
`
`DH69
`B
`
`SHP-77
`
`T
`
`24 h
`
`a’
`C
`(cid:1)0
`C
`.0
`
`0 Sa
`
`’
`
`S CSU1(cid:1)S
`
`a.
`
`3
`Fig.
`100% at
`change
`
`the
`
`of
`Stability
`the
`initiation
`in
`immunoconjugate
`
`of
`
`immunoconjugate
`incubation
`binding
`
`binding
`at 37#{176}C(time
`to SCCL
`cells
`
`lines
`cell
`SCCL
`to four
`three
`0). Results
`from
`in the
`first
`4 h.
`
`was
`individual
`
`by
`analyzed
`experiments
`
`flow
`
`cytometry.
`are
`presented
`
`percentage
`The
`as mean
`± SD.
`
`of binding
`There
`
`is defined
`is no
`significant
`
`as
`
`Tab!
`
`e I
`
`Binding
`
`of
`
`the
`
`immunoconjugate
`
`to monocytes
`
`befor
`
`e and
`
`after
`
`rIFN-y
`
`incubation”
`
`Before
`
`rIFN--y
`
`incubation
`
`After
`
`rIFN-(cid:1)y
`
`incubation
`
`22
`
`% Positive
`
`±
`
`SD
`
`MFI
`
`±
`
`SD
`
`% Positive
`
`±
`
`SD
`
`of
`
`22, F(ab’)2
`
`immunoconjugates
`
`85.2
`84.7
`92.1
`85.8
`
`± 16.7
`± 17.2
`± 7.60
`±
`16.3
`to human
`
`monocytes
`
`MFI
`
`± SD
`
`210.1
`124.6
`188.0
`1 19.6
`before
`
`± 46.7
`± 25.5
`± 85.1
`± 20.9
`and
`
`(2)
`(2)
`(2)
`(2)
`after
`
`18-h
`
`mAb
`F(ab’)2
`22 x Lys3-BN
`mAb
`X Lys3-BN
`F(ab’)2
`a Flow cytometric
`
`analysis
`
`83.5
`70.7
`86.7
`72.3
`the binding
`
`± 2.2
`± 15.6
`± 3.4
`± 10.7
`of mAb
`
`52.0
`29.9
`25.7
`26.7
`of mAb
`
`(2)
`± 26.0
`(2)
`± 14.6
`(2)
`± 0.10
`(2)
`± 8.57
`22, and two
`
`rIFN--y
`
`incubation.
`
`MFI
`
`increased
`
`significantly
`
`after
`
`rIFN--y
`
`incubation.
`
`results
`The
`presented
`are
`cells
`donors
`different
`among
`ied
`greatly
`be
`cells
`could
`lysed
`cell
`lysis was
`primarily
`ual
`donor.
`The
`greatest
`ratio
`of 100:1.
`Pilot
`erate
`cytotoxicity.
`
`of monocyte-mediated
`in Fig.
`5. Since
`in each
`experiment,
`donors.
`For
`each
`by monocytes
`dependent
`lysis was
`experiments
`Increasing
`
`SCCL
`from
`var-
`of
`
`of
`cytotoxicity
`came
`effector
`cells
`the
`potency
`the
`cytotoxic
`line,
`>80%
`SCCL
`cell
`donors.
`Tumor
`from
`some
`in each
`individ-
`on E:T
`ratio
`consistently
`achieved
`at an E:T
`using
`4-h
`assays
`showed
`mod-
`incubation
`time
`from
`4 h to
`
`the
`
`18
`
`h resulted
`
`in
`
`a higher
`
`percentage
`
`of
`
`tumor
`
`cell
`
`lysis.
`
`We
`
`also
`
`-.--
`-0--
`
`Experiment
`Experiment
`
`1
`
`2
`
`150
`
`100
`
`0
`
`0 0
`
`50
`
`1
`
`10
`
`100
`
`1000
`
`10000
`
`100000
`
`Immunoconjugate
`
`concentration
`
`(ng/mI)
`
`concentrations
`of different
`Effect
`4
`Fig.
`x 22 on cytotoxicity
`of 5tCr-labeled
`at
`an E:T
`ratio
`of
`100:1.
`Results
`cell
`lysis
`by monocytes
`achieved
`defined
`as
`100%.
`
`Lys3-BN
`immunoconjugate
`of
`monocytes
`cells
`by
`human
`SHP-77
`two
`experiments
`are
`shown.
`Tumor
`of
`with
`25 p.g/mI
`immunoconjugate
`was
`
`was
`toxicity
`monocytes
`vated
`Results
`cells.
`tumor
`imum
`concentrations.
`the maximum
`
`of
`cell
`Since
`binding
`
`by
`determined
`effector
`as
`such
`two
`lysis
`
`acti-
`using
`assay
`a dose-response
`target
`as
`cells
`cells
`and
`SHP-77
`4. Max-
`in Fig.
`assays
`are
`presented
`range
`of
`a wide
`was
`observed
`over
`flow
`cytometry
`analysis
`indicated
`that
`the
`immunoconjugate
`to SCCL
`cells
`
`the
`of
`
`at
`occurred
`immunoconjugate
`
`concentrations
`in most
`
`25
`above
`cytotoxicity
`
`jig/mI,
`assays.
`
`we
`
`used
`
`25
`
`p.g/ml
`
`could
`donor
`from
`monocytes
`that
`cell
`SCCL
`against
`potency
`cytotoxic
`by monocytes
`better
`be
`could
`cell
`line
`another
`donor.
`to monocytes
`but
`resistent
`differ-
`was
`studied
`under
`cytotoxicity
`Monocyte-mediated
`a typical
`The
`in each
`experiment.
`result
`of
`assay
`ent
`conditions
`in
`Fig.
`6.
`Incubating
`target
`cells
`with
`experiment
`is
`shown
`in
`the
`absence
`of
`immunoconjugate
`re-
`activated
`monocytes
`tumor
`cell
`lysis,
`depending
`on
`of
`suited
`15-60%
`in
`parental
`antibody,
`mAb
`of
`The
`addition
`did
`22,
`In the
`presence
`immunoconjugate,
`lysis.
`increase
`the
`is
`increased
`50-95%.
`This
`increase
`cell
`lysis
`was
`for
`each
`individual
`and
`is
`consistent
`(P <
`
`have
`lines.
`from
`
`dif-
`One
`one
`
`the
`not
`
`donor.
`further
`tumor
`significant
`in each
`
`observed
`ferent
`SCCL
`donor,
`
`same
`the
`different
`killed
`from
`
`to
`
`donor
`
`of
`
`0.05)
`
`
`
`Downloaded from on August 31, 2015. © 1995 American Association for Cancerclincancerres.aacrjournals.org
`
`
`Research.
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1016 Page 5 of 11
`
`(cid:1)
`(cid:1)
`
`
`430
`
`Immunoconjugate
`
`of Lys3-BN
`
`and mAb
`
`22 Can
`
`Induce
`
`Lysis
`
`of
`
`SCCL
`
`DMS273
`
`cells
`
`H69
`
`cells
`
`S
`
`.&
`
`-0---
`
`.-..----
`
`Donorl
`Donor
`
`2
`
`Donor3#
`
`Donor4#
`
`100
`
`80(cid:1)
`
`60(cid:1)
`
`40(cid:1)
`
`20(cid:1)
`
`0 0
`
`SU 0E
`
`2
`
`#{163}
`
`Donor3#
`
`12:1
`
`25:1
`
`50:1
`
`100:1
`
`12:1
`
`25:1
`
`50:1
`
`100:1
`
`H345
`
`cells
`
`SHP-77
`
`cells
`
`S
`
`-0(cid:1)---
`
`________
`
`Donorl
`
`Donor
`
`100
`
`80
`
`60
`
`40
`
`20
`
`100
`
`0 0
`
`.(cid:1).
`
`0U
`
`0
`
`E
`
`I-
`
`S
`
`-0---
`
`#{163}
`
`-..-.--
`
`.--..-.(cid:1).-..-
`
`Donorl
`Donor2
`Donor3
`
`Donor
`4
`Donor5#
`
`12:1
`Effector
`
`#{149}
`25:1
`to
`
`50:1
`target
`
`100:1
`cell
`
`ratIo
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`0 0
`
`SU 0 E
`
`I-
`
`Donorl
`
`Donor
`
`2
`
`Donor3#
`
`#{149}80
`
`60
`
`4(cid:1)
`
`.(cid:1),
`
`:;
`
`U
`
`E20
`
`,(cid:1)
`
`0
`
`#{149}
`12:1
`
`.
`25:1
`
`i
`50:1
`
`I
`100:1
`
`S
`
`___
`
`-#{149}0---
`
`#{163}
`
`SCCL
`of 5tCr-Iabeled
`Cytotoxicity
`Fig.
`in each
`separated
`from different
`donors
`were
`same
`donor
`enhanced
`by
`the monocytes
`the
`of
`in cytotoxicity
`between
`immunoconjugate
`between
`L-BN
`and
`F(ab’),
`of mAb
`22.
`
`by
`cells
`individual
`after
`made
`
`monocytes.
`human
`A 4-h
`assay.
`18-h
`incubation,
`from
`Lys3-BN
`
`Assays
`assay
`was
`subsequent
`and mAb
`
`ratios
`at E:T
`performed
`were
`experiments.
`performed
`in pilot
`using
`assays
`were
`done
`or
`its
`F(ab’)2
`fragments.
`
`22
`
`an
`
`12:1
`from
`ranging
`tumor
`the
`Since
`18-h
`incubation.
`4-h
`assay;
`
`100:1.
`to
`lysis
`cell
`is no
`There
`immunoconjugate
`
`#,
`
`Monocytes
`was
`greatly
`difference
`made
`
`(cid:1),
`
`the
`
`5
`
`individual
`
`assay.
`
`The
`
`sis
`
`could
`
`be
`
`significantly
`
`sive
`
`amounts
`
`of
`
`either
`
`immunoconjugate-induced
`0.05)
`(P <
`unconjugated
`
`blocked
`mAb
`
`by
`
`22
`
`SCCL
`adding
`Lys3-BN
`
`or
`
`ly-
`cell
`exces-
`
`in
`
`assay.
`with
`
`1 mg/mI
`
`of
`presence
`The
`each
`immunoconjugate-induced
`interfere
`potency
`the
`We
`also
`compared
`not
`shown).
`nonstimulated
`and
`between
`rIFN-y
`stimulated
`is
`shown
`in Fig.
`the
`same
`donor.
`The
`result
`rIFN-(cid:1)y
`stimulation
`a significantly
`higher
`0.05)
`in different
`E:T ratios.
`was
`(P <
`
`human
`tumor
`of
`
`IgG
`cell
`tumor
`monocytes
`7. Monocytes
`cytotoxic
`no difference
`
`did
`lysis
`cell
`
`not
`(data
`lysis
`from
`after
`potency
`in the
`
`with
`
`whole
`
`had
`
`There
`
`bated
`tumor
`amounts
`(data
`
`with
`cell
`
`target
`lysis
`either
`of
`shown).
`
`not
`
`cells.
`could
`be
`mAb
`
`the
`Again,
`significantly
`
`immunoconjugate-induced
`blocked
`by
`adding
`
`22
`
`or Lys3-BN
`
`in
`
`each
`
`individual
`
`excess
`
`assay
`
`DISCUSSION
`Recently,
`
`a variety
`
`have
`
`been
`
`shown
`
`in
`
`of
`
`to
`
`bispecific
`
`antibodies
`
`and
`
`immuno-
`
`confer
`
`antitumor
`
`effects
`
`in
`
`vitro
`
`as
`
`major
`to
`
`cytotoxicity
`
`from
`
`immunoconjugate
`
`made
`
`its
`
`toxins
`as
`well
`molecules
`tumor
`
`vivo
`lies
`while
`
`The
`(26-29).
`ability
`in
`their
`simultaneously
`
`cells
`
`advantage
`target
`triggering
`
`these
`bispecific
`of
`selectively
`therapy
`cytotoxicity
`
`by
`
`to
`en-
`
`effector
`dogenous
`immunoconjugates
`fragments
`of mAb22,
`in the
`presence
`of
`in
`vitro.
`
`The
`
`Lys3-BN
`
`In
`
`cells.
`made
`can
`rIFN--y
`
`report
`this
`of Lys3-BN
`effectively
`activated
`
`we
`
`that
`demonstrated
`have
`F(ab’),
`and mAb
`22,
`or
`cells
`induce
`lysis
`of SCCL
`monocytes
`and
`neutrophils
`
`X 22
`
`immunoconjugate
`
`binds
`
`to more
`
`at
`
`SCCL
`
`of
`85%
`than
`cytotoxicity
`while
`that
`suggests
`This
`BN/GRP
`receptors.
`conjugate
`concentrations
`GRP
`receptors
`are
`occupied
`
`concentrations
`cells
`1000-fold
`at
`noted
`is
`lysis
`does
`tumor
`cell
`The
`cytotoxicity
`is particularly
`to a certain
`
`not
`observed
`important
`extent
`
`lower
`require
`at
`
`p.g/ml,
`25
`above
`concentrations.
`saturation
`low immuno-
`since
`the BN/
`endogenous
`
`by
`
`of
`
`cell
`
`SCCL
`greatly
`lysed
`of
`one
`from
`
`lysis
`among
`neutrophils
`by
`cell
`tumor
`when
`donor
`4 h to
`18
`
`are
`do-
`
`lysis
`the
`It
`
`h.
`
`from
`
`at
`on
`
`incu-
`
`observed
`mAb
`
`22
`The
`
`versus
`results
`in Fig.
`shown
`More
`than
`nors.
`two
`different
`from
`greatly
`increased
`was
`incubation
`time
`of
`
`fragments.
`F(ab’),
`neutrophil-mediated
`of
`varied
`8. Cytotoxic
`potency
`were
`75%
`of
`SHP-77
`cells
`donors.
`The
`percentage
`by
`neutrophils
`from
`assay
`was
`prolonged
`
`the
`
`that
`
`appears
`neutrophils.
`an E:T
`ratio
`their
`cell
`neutrophils
`
`are
`
`cells
`H69
`percentage
`The
`100:1.
`Since
`of
`surface
`without
`had
`much
`less
`
`more
`of cell
`neutrophils
`rIFN--y
`cytotoxicity
`
`resistent
`lysis
`
`to
`ranged
`do
`not
`stimulation,
`when
`
`cytotoxicity
`from 17-45%
`express
`Fc-yRI
`nonstimulated
`they
`were
`
`
`
`Downloaded from on August 31, 2015. © 1995 American Association for Cancerclincancerres.aacrjournals.org
`
`
`Research.
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1016 Page 6 of 11
`
`(cid:1)
`(cid:1)
`(cid:1)
`
`
`Clinical
`
`Cancer
`
`Research
`
`431
`
`H69
`
`cslls
`
`100
`
`(cid:1)8o
`
`Cl
`
`C2
`
`T
`
`C3
`
`C4
`
`Cs
`
`Tfln
`
`C3
`
`C4
`
`Cs
`
`DMS273
`
`cells
`
`Cl
`
`C2
`
`T
`
`50.
`
`60
`
`40
`
`0
`
`? .
`
`U
`
`C-
`
`H345
`
`cells
`
`SHP.77
`
`cells
`
`.-
`100
`#{149}0
`
`(cid:1)60
`
`(cid:1)60
`
`U
`
`(cid:1)20
`C-
`
`40
`
`Cl
`
`100
`
`-
`1(cid:1)
`
`#{149}80
`
`(cid:1)60
`
`U
`
`E20
`C-
`
`40
`
`0
`
`C3
`
`CA
`
`C5
`
`Cl
`
`C2
`
`T
`
`C3
`
`C4
`
`CS
`
`Elii(cid:1)iI
`
`C2
`
`I
`
`25 p.g/ml
`assay with
`cytotoxicity
`in each
`incorporated
`They were
`conditions.
`different
`under
`by monocytes
`effects
`without
`with
`immunoconjugate,
`cells
`here.
`incubation
`oftarget
`is presented
`experiment
`of
`100:1.
`One
`typical
`CI,
`without
`activated
`monocytes,
`rIFN-(cid:1)y
`incubation
`of
`target
`with
`itself
`has
`cytotoxic
`effects.
`C2,
`in
`immunoconjugate.
`The
`cytotoxicity
`monocytes
`the
`presence
`the
`target
`cells
`with
`rIFN-y
`activated
`cells
`with
`rIFN-y
`activated
`monocytes
`< 0.05)
`in the
`presence
`of
`immunoconjugate.
`incubation
`target
`C3,
`(P
`activated
`incubation
`of
`target
`cells
`with
`rIFN--y
`monocytes,
`immunoconjugate,
`and
`10-fold
`excess
`of mAb
`C4,
`blocked
`(P < 0.05)
`by
`addition
`of
`excessive
`amounts
`of mAb
`22. CS,
`incubation
`of
`target
`cells
`with
`rIFN-(cid:1)y
`the
`and
`10-fold
`excess
`of Lys3-BN.
`The
`cytotoxicity
`significantly
`blocked
`0.05)
`by
`the
`addition
`<
`
`cells
`
`of
`of
`
`was
`
`(P
`
`of
`
`of
`
`The
`
`Comparison
`6
`Fig.
`immunoconjugate
`monocytes.
`immunoconjugate.
`monocytes
`was
`and mAb
`22,
`but
`22. The
`cytotoxicity
`activated
`monocytes,
`excessive
`amounts
`
`of cytotoxic
`an E:T
`ratio
`and
`immunoconjugate
`incubation
`T,
`significantly
`higher
`no
`immunoconjugate.
`was
`significantly
`immunoconjugate,
`of L-BN.
`
`of
`
`no
`
`in
`
`to
`delivery
`antibody
`and
`BN/GRP,
`the
`approach
`Compared
`with
`limited.
`described
`the
`immunotherapy
`onists,
`antagonists
`advantages.
`First,
`some
`have
`been
`shown
`to partially
`inhibit
`and
`less
`effectively
`vitro
`degraded
`by
`serum
`proteases
`The
`conjugation
`of
`these
`(16).
`noglobulin
`should
`greatly
`retard
`Lys3-BN
`used
`in our
`study
`is an
`the
`immunoconjugate
`thus made
`effect
`on
`the
`growth
`of
`SCCL
`immunoconjugate
`made
`between
`ceptor
`and
`have
`22 will
`mAb
`inhibiting
`growth
`SCCL
`the
`of
`effector
`cells
`to SCCL
`targets.
`BN/GRP
`receptors
`on
`the
`SCCL
`quickly
`upon
`binding
`BN/GRP
`(24).
`tor may
`be disadvantageous
`the
`for
`loss
`onist
`alone
`because
`the
`active
`of
`the
`loss
`of
`inhibitory
`effect
`on SCCL
`the
`gate
`we
`constructed
`can
`greatly
`retard
`and
`BN/GRP
`receptors
`is capable
`of
`inducing
`time.
`a relatively
`short
`Third,
`we
`showed
`jugate
`made
`between
`Lys3-BN
`and
`F(ab’)2
`
`sites
`tumor
`be
`may
`vivo
`in