(12) United States Patent
`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
`U.S. PATENT 13(ITIJM11N'1‘S
`
`6.146.850 A
`6.242.219 BI
`6.274.344 BI
`6.376.217 131
`6.500.648 131
`6.649.742 131
`6.803.210 B2
`6.884.4 18 B1 "
`7.153.932 B2
`
`1172000 Better et a1.
`672001 Be1leret a1.
`872001 Better
`472002 Bettcrctal.
`1272002 Bettcretal.
`1172003 Bettcrctal.
`1072004 Beller
`472005 Shawver et at.
`1272006 Better el al.
`
`4357691
`4357697
`435-697
`4357691
`435-"69.7
`53073873
`435-69.]
`424-1551
`5307326
`
`FOREIGN PATENT DOCUMENTS
`
`AU
`EP
`RF
`W0
`WU
`WU
`
`71—2 172 5788
`0173494
`0239400
`WO 87702671
`WO 89706692
`WU 9370374 1
`
`3' 1989
`371986
`9-' 1987
`5-1987
`771989
`371993
`
`5
`
`[)TllliR PUBLIC‘A’TIONS
`
`Sivan ct al [Cancer Research. 1987. vol. 47. pp. 3169-3173).*
`RudikolT ct al (Proc Natl Acad Sci USA 1982 vol. 79 pp. 1979-
`19831“
`MacCallnm el al. (Journal ofMoleenlaI. Biology. 1996. vol. 262. pp.
`732-745)“
`Pascalis ct a] (Journal of Immunology. 2002. vol. 169. pp. 3076-
`3084).*
`Vajdos El a1. (Joumal of Molecular biology. 2002. vol. 320. pp.
`415—4283“
`
`Holm et al (Molecular Immunology. 2007. vol. 44. pp. 10'75—1084).’a
`Chen et al. (Journal of Molecular Biology. 1999. vol. 293. pp. 865-
`8811*
`“711 et a]. (Journal of Molecular Biology. 199.9. vol. 294. pp. 151—
`162“
`
`Cassel et al (Biochemical and Biophysical Research Communica—
`tions. 2003. vol. 307. pp. 198-2051“
`Batra et al.. “Antitumor activity in mice of an iimnunotoxin made
`with anli—lransl'errin
`receptor
`and a
`recombinant
`form of
`Pseudomonas exoloxin.”Pmr.'. Nat-1. Arrrm’. 51-7.. 86:8545—8549. 1989.
`
`(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
`
`4-"1968 Shirael't'
`471977 Schuursctal.
`771983 I.ilmanel a1.
`371989 Cabillyeta].
`1271989 Lamb-0.11 el al.
`271992 Ilustonetal.
`1271994 Bernhan'l et a].
`571995 Bernhard eta].
`571996 Bacus
`1171996 “’elsetal.
`1271996 King el al.
`471997 Bettcrctal.
`771997 Carter
`771997 Gillies ......
`471998 Bettcrctal.
`571998 Bcttcrctal.
`1171998 Bellerelal.
`1271998 Better eta].
`371999 llustonetal.
`1172000 Bct'tcrctal.
`
`.
`.
`.
`.
`
`
`..
`
`.
`.
`
`
`
`
`
`
`4237272
`43575
`43577.91
`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
`
`Page 2
`
`OTHER PUBUCA'I'IONS
`
`Batra et al.. “Single-chain immunotoxins directed at the human
`transferrin receptor containing Pseudomonas exotoxin A or diphthe-
`ria toxin: anti—TFR(Fv)—PF.4U and DT388—anli—TFR(FV).” Mai. Ce”.
`Bioi.. 112200-2205. 1991.
`Bird et al.. “Single-chain antigen-binding proteins." Science.
`242:423—426. I991.
`Bjorn et al.. “Evaluation ol'Monoclonnl Antibodies for the Develop—
`ment of Breast Cancer trmnunotoxins.“ Cancer Research. 4511214-
`1221.l985.
`Burgess et al.. “Possible dissociation of the heparin-binding and
`mitogenic activities of heparin-binding (acidic fibroblast) growth
`factor-1
`from its
`receptor-binding activities by site-directed
`mutagenesis of a single lysine residue.” .i Ceii Biat. l 1 |:2| 29—2 I 38.
`1990.
`Chaudhary et al.. “A recombinant immunotoxin consisting of two
`antibody variable domains fused to Pseudolnonas exotoxin." Nature.
`339: 394—397. 1989.
`Ciard ielto et al.. “Differential expression of epidermal growth factor-
`related proteins in human colorectal tumors.“ Prec. Nari. Acad. Sci.
`USA. 88277923796. 199].
`Ciardielto et al.. “Expression of cripto. a Novel Gene of d1e Epider-
`mal Growth Factor Gene I-‘amily, Leads to in Vitro Transformation of
`a Norma] Mouse Mammary Epithelial Cell Line.“ cancer Research.
`51210514054. I991.
`Ciccodicola et a] .. “Molecular characterization ofa gene ofthe ‘EGl:
`family‘
`expressed
`in
`undifferentiated
`human N'I'ERAZ
`leralocarcinorna cells.” Enrirol. 8: 198L199 1. I989.
`Coussens et al.. ”I'yrosine Kinase Receptor with Extensive Homol-
`ogy to EGI-' Receptor Shares Chromosomal Location with neu
`Oncogene." Science. 230d l32-l 139. [985.
`Davis et al.. “Single Chain Antibody (SCA) encoding Genes: One
`Step Construction and Expression In Lukaryotic Celts.“ Biotechnor"
`03}: 92165—169. 1991.
`Dilllnan. Ann. inst. Med. 11:592—603. I989.
`Engert et al.. Leukemia Research. 15:10'r'6-1086. 1991.
`l--'itzer-Schiller. The Washington Post, Jan. 19. pp. D3. 1993.
`Gillies and Wesolowski. “Ant igen binding and biological act ivities of
`engineered mutant chimeric antibodies with human tumor speCilici—
`ties.” Harri. Antiboa'. iiyhr‘idornas. 1:47-54. 1990.
`Hancock el al.. “Monoclonal Antibody against the c—er'iJB—Z Protein
`Enhances
`the Cytotoxicity of cis—Diamminedichloroplatinum
`against Human Breast and Ovarian ’t'umor Cell Lines.“ Cancer
`Research. 51:4575-4580. 1991.
`Harlow et al.. Antibodies: A laboratory Manual. Cold Spring Harbor
`Press. Cold Ilarbor Spring, pp. 72-77". 92-91 128-135. 141-157’.
`1988.
`Hoogenboom et al.. “Targeting of tumor necrosis factor to tulnor
`cells: secrtflion by myeloma cells ofa genetically engineered anti-
`body-tumor necrosis
`factor hybrid molecule.” Biochinn'ca et
`Biquhysicc Acta. 1096: 345-354. 1991.
`Hudziak et al.. “p135 HERZ monoclonal antibody has antiprolifera—
`tive effects in vitro and sensitizes human breast cells to tumor necro-
`sis factor.“ Maiecniar' and (‘eihriar' Biology 911165-1139. 1989.
`King et al.. “Amplification of a Novel v—erbB—Relzued Gene in a
`Human Mammary Carcinoma.” Science. 229:974—976. 1985.
`
`Langton et al.. “An Antigen lmmunologically Related to the External
`Domain ofgp l 85 is Shed t'rom Nude Mouse Tumors Overexpressing
`the c-er‘bB-Z (lIer-Zr'nen] Oncogene.“ Cancer Research. 51:2593-
`2598. 199].
`[alas et al. "Transfonning growth factor alpha: mutation of aspartic
`acid 47 and [amine 48 reslllts in different biological activities." Moi.
`Ceii. Biol. 8:124'i-1252. 1988.
`Maier et al.. "Requirements for d1e Internalization of a Murine
`Monoclonal Antibody Directed against the HER—2-"nen Gene Product
`c-er'bB-Z.“ C‘ancer'Research. 515361-5369. 1991.
`McKenzie et al.. “Generation and characterization of monoclonal
`antibodies specific for the human neu oncogene product. plSS.”
`Oncogene. 41:543—548. 1989.
`O‘Ilare et al.. “Cytotoxicity ofa recombinant ricin-A-chain fusion
`protein containing a proteolytically—cleavable spacer sequence.”
`FEBS Lett. 273:200—204. 1990.
`Rosenblum et al.. "An antimetanoma in'nnunotoxin containing
`recombinant human tumor necrosis
`factor:
`tissue disposition.
`phannacokinetic. and therapeutic studies in xenografl models." Cen—
`cer' imnrttttoi imrnttnother'npy. 40:322—328. 1995.
`Rosenbtum et al.. “Antibody-mediated delivery of tumor necrosis
`factor (TN—F—alpha): improvement of cytotoxicity and reduction of
`cellular resistance.” Cancer Communications. 3: 2] —27. I99].
`Roy et al.. “Anti-MY9-blocked-ricin: an immunotoxin for selective
`targeting of acute myetoid leukemia cells.“ Blood. 712404-2412.
`1991.
`Schecter et al.. “the neu Gene: an erbB-llomotogous Gene Distinct
`from and Unlinked to the Gene Encoding the E61: Receptor." Sci—
`ence. 29:976—978. 1985.
`$111M et al.. “A v—erbB-Ielat'ed prolooncogene. c—erbB—Z. is distinct
`from the c-erbB-1.-'epidermal growth factor-receptor gene and is
`amplified in a human salivary gland adenocarcinoma.“ Proc. Nari.
`Acaci. Sci. USA. 82:6497—50I. I985.
`Sivam et al.. “Immunotoxins to a Human Melanoma-associated Anti-
`gen: Comparison of Gelonin with Ricin and Other A Chain Conju-
`gates." Cant-er". Renew-oh. 4?:3169—3l73. 1987.
`Stripe and Barbieri1 “Ribosome—inactivating proteins up to date."
`FEBS Letters. 19511-8. 1986.
`Stripe el al.. "Gelonin. a New Inhibitor of Protein Synthesis. Non—
`toxic to Intact Cells.”.rl. Biol. Chem. 2252694746953. [980.
`Tan et al.. “Studies of aglycosylated chimeric mouse-human lgG.
`Role of carbohydrate in the stnlcture and effector fianctions mediated
`by the human lgG constant region.“ .i. inmnnraiq [43:2595—2601.
`1989.
`'t'ecce et at..Anticancer Research. 1015a): 1454. Abstract 329. 1990.
`Thorpe. "Monoclonal antibodies: clinical and regulatory issues.”
`fiends in Biotechnoi.. 1 1240—42. 1993.
`'t'itt et al.. “An Assay "t'hat Predicts d1e Ability of Monoclonal Anti-
`bodies to l-'orm Potent Ricin A Chain-containing Irrtmunotoxins."
`Cancer Research. 48:] 1 19—1 123. I988.
`Vitetta et al.. “Redesigning nature‘s poisons to create anti-tumor
`reagents." Science. 238:1098-1104. 1989.
`Waidmann. "Monoclonal antibodies in diagnosis and therapy." Sci—
`ence. 252155741562. [99].
`Wels etal...i. C'er‘t'. Biochem.15E:123. 1991.
`
`’5 cited by examiner
`
`IMMUNOGEN 2047, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`IMMUNOGEN 2047, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`US. Patent
`
`Jul. 13, 2010
`
`Sheet] 0132
`
`US 7,754,211 132
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`Phigenix v. Immunogen
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`IPR2014-00676
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`Jul. 13,2010
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`U.S. Patent
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`Jul. 13, 2010
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`Phigenix v. Immunogen
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`IMMUNOGEN 2047, pg. 6
`Phigenix v. Immunogen
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`US. Patent
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`Jul. 13,2010
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`IPR2014-00676
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`US. Patent
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`Jul. 13,2010
`
`Sheet 6 of 32
`
`US 7,754,211 82
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`Phigenix v. Immunogen
`IPR2014-00676
`
`

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`US. Patent
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`Jul. 13, 2010
`
`Sheet 7 of 32
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`Phigenix v. Immunogen
`IPR2014-00676
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`U.S. Patent
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`Jul. 13, 2010
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`Sheet 8 of 32
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`US 7,754,211 132
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`IMMUNOGEN 2047, pg. 10
`Phigenix v. Immunogen
`|PR2014—00676
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`IMMUNOGEN 2047, pg. 10
`Phigenix v. Immunogen
`IPR2014-00676
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`US. Patent
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`Jul. 13,2010
`
`Sheet 9 of 32
`
`US 7,754,211 32
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`
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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
`
`II“
`
`GILOHIH STD:
`
`E “6 TI! 155 ' Cd
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`!fflxlrfaf/Ir/flr/Jrl
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`fizz/)r/r/l/tll
`
`15
`
`i
`
`I
`
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`
`“flu...
`
`10
`
`stBB—PBEL CLONE f
`
`0.25
`
`0.20
`
`nov&
`
`5nu5.0JJ9.0.0o0a
`
`
`
`IVar/f/f/JflI/III/lfjclliIhiidI/Jn/Jr/‘f/l/Jflllnflifllfiilflrd
`
`0.30
`
`
`
`.flffif/f/f/er/Jrluflr/Jfl.
`
`l/f/f/f/f/f/f/fxfl;
`
`y/f/lfl/fflflr/‘f/fé
`
`filly/fill")?
`
`Ill/’2’}.
`
`yf/Ir/f/f/Jfia
`
`.332.339m.um.u.....vfi.p.uo¥..uwo&um
`
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`uuuuuuuuuuuuuu
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`
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`
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`
`[m] cnmnm :19.
`
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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
`
`l
`
`100
`10
`TAD zso-Gdonin (nyml)
`
`-I- MDA—MB-IJI
`
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`
`—.- SKBfi-S
`
`lmplms
`1m
`
`Imam)
`
`4.11131!!!
`
`120
`
`I110
`
`80
`
`61}
`
`‘0
`
`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
`
`o EACH-250
`
`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