`US 7,754,211 32
`(10) Patent N0.:
`Rosenblum et al.
`Jul. 13, 2010(45) Date of Patent:
`
`
`USOO775421 132
`
`(54)
`
`(751
`
`IMMUNOTOXINS DIRECTED AGAINST
`(T-ERBB-2(HER-27NEU) RELATED SURFACE
`ANTIGEN S
`
`Inventors: Michael Rosenhlum. Sugar Land. TX
`(US); Laura K. Shawver. San
`Francisco, CA (US)
`
`(73) Assignee: Research Development Foundation.
`Carson City. NV (US)
`
`( ‘1‘ ) Notice:
`
`Subject to any disclaimer. the term ol‘lhis
`patent is extended or adjusted under 35
`U.S.C. 154(1)) by 731 days.
`
`(21) Appl.No.: 107964,]95
`
`(22) Filed:
`
`Oct. 13. 2004
`
`(65)
`
`Prior Publication Data
`
`US 200570]63774 Al
`
`Jul. 28. 2005
`
`Related U.S. Application Data
`
`(60) Division of application No. 097320.156. filed on May
`26. 1999. now abandoned. which is a continuation-in-
`part ot'application No. 087404.499. filed on Mar. 17.
`1995. now abandoned. which is a contimlation—in—part
`01‘ application No. 087300.082. [iled on Sep. 2. 1994.
`now abandoned, which is a continuation of application
`No. 087164.638. liled on Dec. 9. 1993. now abatt-
`doned. which is a continuation of application No.
`077867.728, filed 011 Apr. 10. 1992. now abandoned.
`
`(51)
`
`Int. Cl.
`A61K 39/395
`(52] U.S.CI.
`
`(2006.01)
`4247'134.l;4247135.1:424-7138.]:
`424041.124247143.l;4247155.l:4247183.]:
`53073873: 53073877: 53073881: 530738885:
`53073913
`
`(58) Field ofClassification Search
`See application file for complete search history.
`
`None
`
`(56)
`
`References Cited
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`4357691
`4357697
`435-697
`4357691
`435-"69.7
`53073873
`435-69.]
`424-1551
`5307326
`
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`AU
`EP
`RF
`W0
`WU
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`71—2 172 5788
`0173494
`0239400
`WO 87702671
`WO 89706692
`WU 9370374 1
`
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`(Continued)
`
`Primary Examiner Karen A Canella
`(74) Aflomejz Agent. or Firm—Fulbright & Jaworski LLP
`
`(57)
`
`ABSTRACT
`
`Novel immunotoxins and methods of treating neoplastic dis—
`eases are provided. More specifically, ilmnunotoxins com—
`prised conjugation ot' a cerbB—Z targeting moiety and a cell
`growth modulator are provided. These inununotoxins specifi-
`cally and selectively kill ltunor cells that over-express the
`c—erbB —2 protein. The novel immunotoxins would be usefill in
`treating human mammary carcinomas. human ovarian carci—
`nomas.
`lung carcinomas. gastric tumors. salivary gland
`adenocareinomas. and colon adenocarcinomas.
`
`10 Claims, 32 Drawing Sheets
`
`IMMUNOGEN 2047, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`3.376.110 A
`4.016.043 A
`4.391.904 A
`4.816.567 A
`4.888.415 A
`5.091.513 A
`5.376.546 A
`5.416.202 A
`5.514.554 A
`5.571.894 A
`5.587.458 A
`5.621.083 A
`5.648.237 A *
`5.650.150 A
`5.744.580 A
`5.756.699 A
`5.837.491 A
`5.851.802 A
`5.877.305 A *
`6.146.631 A
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`4237272
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`53073873
`53073919
`53073873
`..... 4357199
`5367232
`43577.23
`.. 53073873
`53073873
`.
`.. 53073919
`
`435769.]
`.. 42471341
`5307377
`.. 5367234
`35769.1
`4357697
`536723.53
`42471831
`
`IMMUNOGEN 2047, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`
`
`US 7,754,211 B2
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`Page 2
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`’5 cited by examiner
`
`IMMUNOGEN 2047, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
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`IMMUNOGEN 2047, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`
`
`US. Patent
`
`Jul. 13, 2010
`
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`
`US 7,754,211 132
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`Sheet 6 of 32
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`Sheet 8 of 32
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`IMMUNOGEN 2047, pg. 10
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`Sheet 9 of 32
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`US 7,754,211 32
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`
`FIG. 9
`
`IMMUNOGEN 2047, pg. 11
`Phigenix v. Immunogen
`IPR2014-00676
`
`IMMUNOGEN 2047, pg. 11
`Phigenix v. Immunogen
`IPR2014-00676
`
`
`
`U.S. Patent
`
`Jul. 13,2010
`
`Sheet 10 of 32
`
`US 7,754,211 32
`
`I l I Il I I I I I I II ll II II
`
`U cuLTUlI sur
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`GILOHIH STD:
`
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`
`stBB—PBEL CLONE f
`
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`
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`
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`
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`
`.flffif/f/f/er/Jrluflr/Jfl.
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`
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`
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`
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`
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`
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`
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`
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`
`FIG. 10
`
`IMMUNOGEN 2047, pg. 12
`Phigenix v. Immunogen
`|PR2014—00676
`
`IMMUNOGEN 2047, pg. 12
`Phigenix v. Immunogen
`IPR2014-00676
`
`
`
`
`
`
`
`U.S. Patent
`
`Jul. 13, 2010
`
`Sheet 11 0132
`
`US 7,754,211 32
`
`FIG.11
`
`140ann— 87km»
`
`48kD>~
`
`331(1)»-
`
`28kD>~
`
`IMMUNOGEN 2047, pg. 13
`Phigenix v. Immunogen
`|PR2014—00676
`
`IMMUNOGEN 2047, pg. 13
`Phigenix v. Immunogen
`IPR2014-00676
`
`
`
`US. Patent
`
`Jul. 13, 2010
`
`Sheet 12 of 32
`
`US 7,754,211 32
`
`1.5
`
`Absorbanc:
`(405 nm)
`
`'
`
`O
`
`0.5
`
`10'
`
`10°
`
`10'
`
`19'
`
`10'
`
`10'
`
`Concentration (x 10’ 31311111)
`
`FIG. 12
`
`—I- TAhISU
`
`+ TAB BOgemmn
`"'0' ZME
`
`IMMUNOGEN 2047, pg. 14
`Phigenix v. Immunogen
`|PR2014—00676
`
`IMMUNOGEN 2047, pg. 14
`Phigenix v. Immunogen
`IPR2014-00676
`
`
`
`US. Patent
`
`Jul. 13, 2010
`
`Sheet 13 of 32
`
`US 7,754,211 32
`
`R
`
`B
`
`Displacement 01‘“ I-BACh 250 Binding
`
`Displacement 01‘251-5ACH 250m“ Binding
`
`+ SAC)! 2501136 ‘1.Bound
`
`—i-* MouselgGu
`—.‘—' BAG!) 250
`
`ID
`
`100
`
`0.0
`
`0. I
`
`l
`
`I a
`
`ICO
`
`Anllha dls: 131M111)
`
`0.0
`
`I1 I'
`
`I
`
`Antibodies {pgfml}
`
`FIG. 13
`
`IMMUNOGEN 2047, pg. 15
`Phigenix v. Immunogen
`|PR2014—00676
`
`IMMUNOGEN 2047, pg. 15
`Phigenix v. Immunogen
`IPR2014-00676
`
`
`
`US. Patent
`
`Jul. 13, 2010
`
`Sheet 14 of 32
`
`US 7,754,211 32
`
`100
`
`Elm amass maul-54.59 pp:
`111.0 ire/ll 311131—250
`ICESOI-fim all
`00.! 1mm Elma-250 H.150! #105 p)!
`00.01 will 3501-250 18501-515 pH
`
`
`
` PercentCytotoxicity g
`
`10‘13
`
`10—11
`
`10’10
`
`10—9
`
`10—3
`
`Conjugate Conc..M01ar
`
`FIG. 14
`
`IMMUNOGEN 2047, pg. 16
`Phigenix v. Immunogen
`|PR2014—00676
`
`IMMUNOGEN 2047, pg. 16
`Phigenix v. Immunogen
`IPR2014-00676
`
`
`
`US. Patent
`
`Jul. 13, 2010
`
`Sheet 15 of 32
`
`US 7,754,211 32
`
`43» 0.0] "3/1111
`
`+ lughnl
`
`-+- 05 ugfml
`
`-I- 0.1 ugfml
`
`% of
`Carmel
`
`~0- OJDS ugfnfl
`
`IMMUNOGEN 2047, pg. 17
`Phigenix v. Immunogen
`|PR2014—00676
`
`IMMUNOGEN 2047, pg. 17
`Phigenix v. Immunogen
`IPR2014-00676
`
`
`
`US. Patent
`
`Jul. 13,2010
`
`Sheet 16 of 32
`
`US 7,754,211 El
`
`120
`
`I00
`
`80
`
`50
`
`40
`
`20
`
`9301'
`Comm]
`
`D
`
`0.10
`
`I
`
`1
`
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`
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`
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`
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`
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`
`lmplms
`1m
`
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`
`4.11131!!!
`
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`
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`
`80
`
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`
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`
`213
`
`D
`
`1000
`
`I
`
`FIG. 16
`
`1000
`
`mo
`10
`TA!) l'rO-Gclmin (nyml)
`“captors
`am:
`150.1130
`Jasmin
`1.000.000
`
`*- MCF-T
`4- OVCAR—J
`+ MBA-M345}
`-- smut-3
`
`IMMUNOGEN 2047, pg. 18
`Phigenix v. Immunogen
`|PR2014—00676
`
`IMMUNOGEN 2047, pg. 18
`Phigenix v. Immunogen
`IPR2014-00676
`
`
`
`U.S. Patent
`
`Jul. 13,2010
`
`Sheet 17 0f 32
`
`US 7,754,211 32
`
`3%
`ca
`:2
`
`:00 a
`
`10.0
`
`1 o
`
`O I
`
`l BACH-ZSUrGel
`
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`
`9
`
`.
`
`‘0
`
`50
`HOURS
`
`60
`
`170
`
`50
`
`90
`
`1013
`
`FIG. 17
`
`IMMUNOGEN 2047, pg. 19
`Phigenix v. Immunogen
`|PR2014—00676
`
`IMMUNOGEN 2047, pg. 19
`Phigenix v. Immunogen
`IPR2014-00676
`
`
`
`U.S. Patent
`
`Jul. 13,2010
`
`Sheet 18 0f 32
`
`US 7,754,211 32
`
`2.00
`
`1.75
`
`[:l BACH~250
`
`1.50
`
`@BACH—afio/RG
`
`
`
`
`
`RATIO 53CDt-hu-n-Lnuit:'C3L310a TISSUE:BLOOD
`
`FIG. 18
`
`IMMUNOGEN 2047, pg. 20
`Phigenix v. Immunogen
`|PR2014—00676
`
`IMMUNOGEN 2047, pg. 20
`Phigenix v. Immunogen
`IPR2014-00676
`
`
`
`US. Patent
`
`Jul. 13, 2010
`
`Sheet 19 of 32
`
`US 7,754,211 32
`
`2.00
`
`1.75
`
`BEACH-250
`
`@ BACH-EEO/FIG
`
`
`
`RATIO TISSUE:BLDUD
`
`IMMUNOGEN 2047, pg. 21
`Phigenix v. Immunogen
`|PR2014—00676
`
`IMMUNOGEN 2047, pg. 21
`Phigenix v. Immunogen
`IPR2014-00676
`
`
`
`U.S. Patent
`
`Jul. 13, 2010
`
`Sheet 20 0f 32
`
`US 7,754,211 32
`
`2.00
`
`:35
`
`Dam—250
`
`flaw—2500110
`
`1.50
`
`1.25
`
`1.00
`
`0.75
`
`
`
`BLOODRATIO TISSUE:
`
`0.50
`
`0.25
`
`0.00
`
`FIG. 20
`
`IMMUNOGEN 2047, pg. 22
`Phigenix v. Immunogen
`|PR2014—00676
`
`IMMUNOGEN 2047, pg. 22
`Phigenix v. Immunogen
`IPR2014-00676
`
`
`
`U.S. Patent
`
`Jul. 13,2010
`
`Sheet 21 of 32
`
`US 7,754,211 32
`
`
`
`
`
`TISSUE:BLOODRATIO
`
`2.00
`
`1.75
`
`1.5
`
`1.25
`
`1.00
`
`E] EACH-250
`
`@ BACH-ZSO/RG
`
`
`
`IMMUNOGEN 2047, pg. 23
`Phigenix v. Immunogen
`|PR2014—00676
`
`IMMUNOGEN 2047, pg. 23
`Phigenix v. Immunogen
`IPR2014-00676
`
`
`
`U.S. Patent
`
`Jul. 13,2010
`
`Sheet 22 of 32
`
`US 7,754,211 32
`
`[:1 EACH-250
`2'00 [
`1.75
`r 1
`BACH-ESG/HS
`
`1.25?
`
`
`1
`1.00:
`mil
`
`T18RATIO
`
`
`
`
`
`025 ,~
`
`
`
`:'
`'I
`.‘I‘
`a"
`I'
`‘
`'4'.’
`-‘
`4 HR
`12 HR
`95 HR
`34 HR
`
`0.503-
`
`0'00
`
`43 HR
`
`72 HR
`
`HOURS
`
`F1(§.22
`
`IMMUNOGEN 2047, pg. 24
`Phigenix v. Immunogen
`|PR2014—00676
`
`IMMUNOGEN 2047, pg. 24
`Phigenix v. Immunogen
`IPR2014-00676
`
`
`
`U.S. Patent
`
`Jul. 13,2010
`
`Sheet 23 0f 32
`
`US 7,754,211 32
`
`"' PBS
`
`«0- TAb 250 + Gclonin
`
`'4'" TA!) ZSO-Gclonin
`
`4
`
`6 6101214161820222426 283032343638 404244464850
`
`Day
`
`FIG. 23
`
`IMMUNOGEN 2047, pg. 25
`Phigenix v. Immunogen
`|PR2014—00676
`
`IMMUNOGEN 2047, pg. 25
`Phigenix v. Immunogen
`IPR2014-00676
`
`
`
`U.S. Patent
`
`Jul. 13,2010
`
`Sheet 24 of 32
`
`US 7,754,211 32
`
` 0
`
`5
`
`‘l D
`
`1 5
`
`20
`
`25
`
`30
`
`35
`
`4O
`
`45
`
`50
`
`55
`
`50
`
`FIG. 24
`
`IMMUNOGEN 2047, pg. 26
`Phigenix v. Immunogen
`|PR2014—00676
`
`IMMUNOGEN 2047, pg. 26
`Phigenix v. Immunogen
`IPR2014-00676
`
`
`
`US. Patent
`
`Jul. 13, 2010
`
`Sheet 25 of 32
`
`US 7,754,211 32
`
`212 Link:
`
`(G115:-r Th! Sr: Glr Sn Gly Ln 5:: 5a 51.. 01; Ly: Gly] (5:1 10 M» "009413;“
`
`Thm b'
`
`
`
`.....
`
`‘‘‘‘‘
`
`unlufi's
`
`. 1
`
`(Glyfiiyfihfiiy 5d) Cm '9’” )..........
`
`[Hit].
`
`u”
`
`
`
`IMMUNOGEN 2047, pg. 27
`Phigenix v. Immunogen
`|PR2014—00676
`
`IMMUNOGEN 2047, pg. 27
`Phigenix v. Immunogen
`IPR2014-00676
`
`
`
`US. Patent
`
`Jul. 13, 2010
`
`Sheet 26 of 32
`
`US 7,754,211 32
`
`._..____._.
`LANE
`
`SAMPLE
`
`- A 'I'NF(l7kD)Slandzu-d
`
`A B c 1) E F G H 1 J
`
`* B
`
`Uninduccd stZB bacteria] Iysate
`
`. C
`
`Induced 5Fv23 soluble lysatc
`
`.
`
`|')
`
`Affinity (IMAC) resin prior to elation
`
`sI-‘v23 clunlc from affinity resin - F
`
`- E
`
`Uninduccd sI-‘v23—TNF bacterial lysatc
`
`- G
`
`Induced sl’v23-TNF soluble lysate
`
`. H A [‘finily (IMAC) resin prior to clution
`
`-
`
`°
`
`I
`
`J
`
`stEJ-TNF conjugate from affinity resin
`
`Mulccular weight markers
`
`FIG. 26
`
`IMMUNOGEN 2047, pg. 28
`Phigenix v. Immunogen
`|PR2014—00676
`
`IMMUNOGEN 2047, pg. 28
`Phigenix v. Immunogen
`IPR2014-00676
`
`
`
`US. Patent
`
`Jul. 13, 2010
`
`Sheet 27 of 32
`
`US 7,754,211 32
`
`Coomassie
`
`Anti-5M3 Ab
`
`Anti-TM? Ab
`
`
`
`FIG. 27
`
`IMMUNOGEN 2047, pg. 29
`Phigenix v. Immunogen
`|PR2014—00676
`
`IMMUNOGEN 2047, pg. 29
`Phigenix v. Immunogen
`IPR2014-00676
`
`
`
`U.S. Patent
`
`Jul. 13,2010
`
`Sheet 28 0132
`
`US 7,754,211 32
`
`C‘st23-TNF(His)5
`
`Elst23(His)5
`
`0
`
`250
`
`500
`
`750
`
`1000
`
`Concentration (pH)
`
`FIG. 28
`
`IMMUNOGEN 2047, pg. 30
`Phigenix v. Immunogen
`|PR2014—00676
`
`IMMUNOGEN 2047, pg. 30
`Phigenix v. Immunogen
`IPR2014-00676
`
`
`
`U.S. Patent
`
`Jul. 13, 2010
`
`Sheet 29 of 32
`
`US 7,754,211 32
`
`0.0.590
`
`0.25
`
`0.50
`
`0.75
`
`1.60
`
`1.25
`
`CONCENTRATIomuM)
`
`FIG. 29
`
`IMMUNOGEN 2047, pg. 31
`Phigenix v. Immunogen
`|PR2014—00676
`
`IMMUNOGEN 2047, pg. 31
`Phigenix v. Immunogen
`IPR2014-00676
`
`
`
`U.S. Patent
`
`Jul. 13,2010
`
`Sheet 30 of 32
`
`US 7,754,211 32
`
`H
`
`4—- HER ma;
`
`.3
`
`me
`
`so
`
`ISO
`
`'8
`‘.:
`5o
`
`:3
`
`do
`
`20
`
`o
`m'
`
`adv-SKEW LP
`FPSKBRS HP
`
`ml
`
`10"
`
`10’
`
`W
`
`TNF Concentration
`
`{Uniufml}
`
`FIG. 30
`
`IMMUNOGEN 2047, pg. 32
`Phigenix v. Immunogen
`|PR2014—00676
`
`IMMUNOGEN 2047, pg. 32
`Phigenix v. Immunogen
`IPR2014-00676
`
`
`
`U.S. Patent
`
`Jul. 13,2010
`
`Sheet31 0132
`
`US 7,754,211 132
`
`st23-TNF (His)5
`
`TNF
`
`'
`
`I
`
`%ofControl
`
`10’
`
`103
`
`103
`
`10‘
`
`105
`
`10‘5
`
`Concentration (ulml)
`
`FIG. 31
`
`IMMUNOGEN 2047, pg. 33
`Phigenix v. Immunogen
`|PR2014—00676
`
`IMMUNOGEN 2047, pg. 33
`Phigenix v. Immunogen
`IPR2014-00676
`
`
`
`U.S. Patent
`
`Jul. 13,2010
`
`Sheet 32 of 32
`
`US 7,754,211 132
`
`+ W
`
`+ sfv23fTNF
`
`100
`
`80
`
`so
`
`4o
`
`2010’
`
`10‘
`
`105
`
`10‘
`Concentration
`
`10’
`
`(Unitalml)
`
`10'
`
`10"
`
`FIG. 32
`
`IMMUNOGEN 2047, pg. 34
`Phigenix v. Immunogen
`|PR2014—00676
`
`.9.-I:
`
`o 2
`
`0 3
`
`2
`
`IMMUNOGEN 2047, pg. 34
`Phigenix v. Immunogen
`IPR2014-00676
`
`
`
`US 7,754,21 l 32
`
`1
`[M MUNOTOXINS DIREC’I'ED AGAINST
`C-ERBB-2(HER-2fN EU) RELATED SURFACE
`ANTIGENS
`
`CROSS RlZI’liRliNCli TO RlELA’l‘I il)
`APPLICATION
`
`The present application is a divisional of application Ser.
`No. 0911320, 156 filed May 26, 1999 now abandoned, which is
`a continuation in part of US. patent application Ser. No.
`081404.499. filed Mar. 17, 1995 now abandoned, which is a
`continuation in part of US. patent application Ser. No.
`0811300082. filed Sep. 2. 1994, now abandoned. which is a
`continuation of U .S. patent application Ser. No. 081'1641338.
`filed Dec. 9, 1993, now abandoned, which is a continuation of
`US. patent application Ser. No. 07861728, filed Apr. 10,
`1992, now abandoned.
`BACKGROUND OF THE INVENTION
`
`2
`
`5
`
`if]
`
`15
`
`ing region. exhibiting binding specificity for the c-erbB-Z
`protein and a cell growth modulator, e.g., a toxin or growth
`suppressing reagent. This composition acts as an immuno—
`toxin to specifically target a cell growth modulator to tumor
`cells overcxprcssing the c-erbli-Z protein.
`Thus. in one embodiment ofthe present invention, there is
`provided a new composition oi'inattercomprisiiig a conjugate
`oi'a targeting moiety with binding specificity for the cerbB—2
`protein, e.g., TAb 250 monoclonal antibody, and a cytotoxic
`moiety. The cytotoxic moiety may be a toxin, a cytocida]
`drug, a cytostatic drug. or a biological response modifier. In
`one particular embodiment, the cytotoxic moiety is gelonin.
`Another embodiment of the present invention provides a
`method oftreating a neoplastic condition. e.g.. disease. which
`is characterized by amplification or overexprcssion of the
`c-crbB-Z oncogene. comprising administering a cytocidally
`effective dose of an immunotoxin of the present invention to
`an individual in need of such treatment.
`
`30
`
`60
`
`Finally. another embodiment of the instant invention com—
`prises a method of treating neoplastic cells with an antibody—
`‘l‘Nl: conjugate. Possible target cells include mammary car-
`cinoma cells. ovarian carcinoma cells, ltmg carcinoma cells,
`salivary gland carcinoma cells. gastric tumor cells, colon
`adenocarcinoma cells, and bone marrow leukemia cells. A
`specific example entailing breast carcinoma cells is provided.
`
`ISRIIEI’ DESCRIPTION 0]" THE DRAWINGS
`
`FIG. 1 demonstrates the effects of ZME antibody. TAb 250
`antibody or innnunonconjugates ofTAb 250 and gelonin on
`SKOV—3 cells as measured by l.il.lSA.
`l“ [(i. 2 demonstrates the cytotoxicity of the 'lAb 250 gelo-
`nin construct on SKOV-3 cells.
`
`IMMUNOGEN 2047, pg. 35
`Phigenix v. Immunogen
`IPR2014-00676
`
`SUMMARY OF THE INVENTION
`
`'llie present invention provides a composition comprising a
`conjugate ofa cellular targeting moiety. e.g., an antigen bind-
`
`In still another embodiment ofthe present invention, there
`is provided compositions of matter comprising fusion con-
`strttcts of targeting moieties with binding-affinity for
`c—erbB—2 protein and a cytotoxic moiety. Preferably. the tar—
`geting moiety is an antibody which recognizes an extracellu—
`lar epitope of c—crbB—2. e.g.. TAb 250. and the cytotoxic
`moiety is relatively inert when applied separately from the
`targeting moiety, e.g.. gelonin. In other embodiments of the
`present invention there are provided methods ofextending the
`survival time ofa tumor bearing mammal by administration
`oftargeted toxins o fthe present invention to this mammal and
`also a method of retarding the rate of growth of tumors by
`administering targeted toxins of the present invention. 'fypi-
`cally, the targeted toxins will be targeted by an immunologi—
`cal binding region, e.g._. an antibody binding segment. Addi—
`tionally
`provided
`is
`a
`pharmaceutical
`composition
`‘ comprising an immunotoxin consisting essentially ofa cyto-
`toxic moiety conjugated to a monoclonal antibody. Most pref—
`erably. the antibody is TAb 250 and the cytotoxic moiety is
`gelonin.
`In another embodiment of the instant invention, there is
`provided a conjugate of tumor necrosis factor to an antibody
`exhibiting binding specificity for an extracellular epitope of
`c—erbB—2 protein. The antibody may be an intact filll length
`antibody with either the heavy chain or light chain peptide
`conjugated to tumor necrosis factor. Alternatively, the anti-
`body may hc or a Irv fragment with the toxin linked to either
`the VL or VH peptide. In the preferred embodiment. conjugate
`is a fusion protein between a single chain antibody and tumor
`necrosis factor which is preferably produced by recombi—
`nantly fusing a gene encoding a single chain antibody to a
`gene encoding tumor necrosis factor. One possible sliv is
`scFv—23.
`
`40
`
`45
`
`50
`
`1. Field of the Invention
`The present invention relates generally to the field oftreat-
`ment of neoplastic disease. More specifically. the present
`invention relates to novel innnunoconiugates and their use in
`the treatment of neoplastic disease.
`2. Description of the Related Art
`Neoplastic disease is one ofthe leading causes ofmortality
`and morbidity in the Western World. Neoplastic conditions,
`e.g.. diseases or “cancers”, share at least one characteristic,
`i.e.. the involvement oi'defects in the cellular growth regula—
`tory process. The process by which normal cells are traits-
`forTned into malignant cells has been a subject of intense
`study for decades. More recently. study has focused on the
`role ofoncogenes in the cancer process. Oncogenes are genes
`that have the ability to transform eukaryotic cells so that they
`grow in a manner analogous to tumor cells.
`An oncogene is created when a normal gene or proto-
`oncogene is mutated, rearranged, or amplified. One such
`oncogene is
`the cerbB—Z(HER—21neu) proto—oncogene.
`Hereinafter. this oncogene will be referred to as c-erbB-Z.
`This gene encodes a protein similar to epidermal growth
`factor receptor. Amplification of this proto-oncogenc can
`result in a cascade of cellular events leading to unregulated
`cell growth.
`Antibodies are proteins produced by the immtme system of
`an animal, normally in response to foreign antigens or anti-
`genic determinants. Antibodies bind to the specific antigen to
`which they are directed. The development of specific mono—
`clonal antibodies has provided investigators with a possible
`means of selectively targeting therapeutic agents to cells
`which overcxpress defined antigens.
`Overexpression of the c—erbB—2 proto—oncogene in neo—
`plastic transformation has been postulated. Several types of
`human cancers including some mammary carcinomas and
`some ovarian carcinomas have an amplified c-erbB-Z gene. 5‘
`Moreover. amplification and subsequent overcxprcssion of
`i
`the c—erbB—2 gene has been correlated with poordisease prog—
`nosis. Thus. there exists a great need and desire in this art for
`a method of selectively targeting a chemotherapeutic agent to