United States Patent r19J
`Pastan et al.
`
`111111111111111111111111111111111111111111111111111111111111111111111111111
`US005889157 A
`[11] Patent Number:
`[45] Date of Patent:
`
`5,889,157
`*Mar. 30, 1999
`
`(54] HUMANIZED 83 ANTIBODY FRAGMENTS,
`FUSION PROTEINS, AND USES THEREOF
`
`(75]
`
`Inventors: Ira Pasta n, Potomac; Itai Benbar,
`Rockville; Eduardo A. Padlan,
`Kensington; Sun-Hee Jung, Rock-ville;
`Byungkook Lee, Potomac, al l of Md.
`
`[73] Assignee: T he United States of A merica as
`represented by the Department of
`Health and Huma n Services,
`Washington, D.C.
`
`[ * ] Notice:
`
`Tlie term of tliis palenl shall not extend
`beyond the expiration date of Pat. No.
`5,242,813.
`
`[21J Appl. No.: 331,396
`
`(22) Filed:
`
`Oct. 28, 1994
`
`Related U.S. Application Data
`
`[63] Continuation-in-part of Ser. No. 767,331. Sep. 30, 1991,
`abandoned, which is a conliouation-in-part of Ser. No.
`596,289, Oct. 12, 1990, Pat. No. 5,242,813.
`Int. C l.6
`......................... C07K 16/00; A61K 39/395
`[51]
`[52] U.S. C l ...................................... 530/387.l.; 530/387.3;
`530/387.5; 5301387.7; 530/388.l; 530/388.8;
`424/133.l; 435/328; 435/7.1; 536/23.53
`[58] Field of Search .............................. 530/387.l, 387.3,
`530/387.5, 387.7, 388.1, 388.8, 390.5, 866,
`867; 435/69.1, 69.7, 91.1, 7.1, 328; 536/23.53;
`424/133.l
`
`[56]
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`4,545,985
`4,867,962
`5,242,813
`5,258,498
`
`10/1985 Paslaa el a I. .
`9/1989 Abrams .
`9/ 1993 Pastan el al. .
`11/ 1993 Houston el al. ........................ 530/350
`
`Hartman ct al., E.M.B.O. J. 3: 3023-3030 (1984).
`Hellstrom ct al., Cancer Res., 50: 2183- 2190 (1990).
`Hoess et al., Gene, 128: 43-49 (1993).
`Huston et al., Proc. Natl. Acad. Sci USA, 85:5879-83
`(1988).
`Jones el al., Nature, 321: 522-525 (1986).
`Jones et al., Nature, 321: 522-525( 1986).
`Kondo ct al.,J. Biol. Chem., 263: 9470-75 (1988).
`Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855
`(1984).
`Pai ct al., Proc. Natl. Acad. Sci. USA, 88: 3358- 62 (1991).
`Pastan et al., Cancer Res., 51: 3781-3787 (1991).
`Wawrzynczak et al., Mo!. /111111unol., 29: 213-220 (1992).
`Willingham et al., Proc. Natl. Acad. Sci. USA, 84:
`2474-2478 (1987).
`Brinkmann et al., Bochem. Biophys Acta., 1198:27-45,
`1994.
`Proceedings of the National Academy of Sciences of the
`USA, vol. 88, No. 19, 1 Oct. 1991 Washington, DC, USA,
`pp. 8616-8620, U. Brinkmann et al. 'B3 (Fv)-PE38KDEL,
`a single-chain immunotoxin that causes complete regression
`of a human carcinoma in mice' cited in the application see
`abstract see FIGS. L,2.
`Bioconjugaie Chemistry, vol. 5, No. 4, Jul. 1994 Washing(cid:173)
`ton, DC, USA, pp. 321- 326, XP 000564453 1. Beobar et al.
`'Mutations of two lysine residues in 1be CDR loops of a
`recombinant immunotoxin tbat reduce its sensitivity to
`chemical derivatization.' cited in 1he application see abstract
`see FIG. 1.
`Cancer Research, vol. 53, No. 2, 15 Jaa. 1993 Philadelphia,
`PA, USA, pp. 334-339, P. Friedman et al. · BR96 sFv-PE40,
`a potent singlc-cbain immunotoxio tbat selectively kills
`carcinoma cells.' see abstract see discussion.
`The Journal of Immunology, vol. 152, No. 5, 1 Mar. 1994
`Baltimore, MD, USA, pp. 2377-2384, C. Siegall et al., ' In
`vitro and in vivo cbarac1erization of BR96 sFv-PE40.' see
`abstract.
`
`Primary Examiner-Frank C. Eisenscbcnk
`Attorney, Agent, or Firm- Townsend and Townsend and
`Crew LLP
`
`FOREIGN PATENT DOCUMENTS
`
`[57]
`
`ABSTRACT
`
`93 07286
`
`4/ 1993 WIPO .
`
`OTHER PUBLICATIONS
`
`Bast ei al., .T. Clin. Invest., 68:1331- 1337 (1981).
`Batra et al., J. Biol. Chem. 265: 15198-15202 (1990).
`Batra et al., Proc. Nnrl. Acad. Sci. USA 86: 8545-8549
`(1989).
`Beller et al., Science, 240: 1041-1043 (1988).
`Bird et al., Science, 242: 423-26 (1988).
`Brinkmann et al., Proc. Nat. Acad. Sci. USA 89: 3075-3079
`(1992).
`Chaudhary ct al., Nature, 339: 394-97 (1989).
`Chaudhary et al., Proc. Natl. Acad. Sci. USA 87:9491- 94
`(1990).
`Chaudhary ct al., Proc. Nari. A cad. Sci. USA 87: 1066- 1070
`(1990).
`
`This invention provides for recombinant single cbain anti(cid:173)
`bodies capable of specifically binding to a Lewis1'-related
`carbohydrate amigen and fusion proteins comprising these
`antibodies. More particularly, the invention provides for
`bumaoized chain Fv regions of the monoclonal antibodies
`Bl, B3 and BS and fusion proteins incorporating tbese
`humanized antibod ies. Tbe antibodies may comprise
`humanized variable heavy (V H) chains, humanized variable
`light (VL) chains, or both. The invention also provides for
`DNA sequences encoding the various humanized antibodies.
`In addition, the invemion provides for methods of detecting
`cells bearing a Lewis1' antigen in a parienl and for methods
`of killing or inhibiting the growth of cells bearing a LewisY
`antigen in a patient.
`
`21Claims,17 Drawing Sheets
`
`1 of 62
`
`BI Exhibit 1123
`
`

`

`U.S. Patent
`
`Mar. 30, 1999
`
`Sheet 1of17
`
`5,889,157
`
`PCR (OUGO B3HI + B3H2)
`I
`CA. 370 BP FRAGMENT
`Ndel
`BomHI
`I
`BomHI
`CLONING IN pBR322/BomHI/ AP
`(pULEE I)
`I
`1 - - 1 _v_H _ILi
`'
`'
`
`~deI
`'
`
`BomHl
`I
`
`/
`
`/
`I_,,
`/
`
`'
`
`'
`
`/
`
`/
`
`-
`
`PCR<OLIGO 83L1•83L2)
`I
`111 CA. 370 BP FRAGMENT I
`BomHI
`I
`Hindm
`CLONING IN BR322/Hindm/AP
`(pULEE 2)
`I
`............ _v ____ L _j
`N
`...-BomHI
`-
`--
`/ /
`
`Hindm
`
`_Hindm
`
`_,,..--
`
`-
`
`-
`
`P(T7)
`
`Ndel
`
`Hindm
`
`pULEE 3
`
`blo
`
`SITE-DIRECTED MUTAGENESIS
`
`P(T7)
`
`N el
`
`Hindm
`
`pULI 1
`
`REPLACE PE40 BY PE38KDEL
`FROM pRK79K
`PE40
`
`VH
`
`VL
`
`P(T7)
`
`FIS. IA.
`
`blo
`
`bla
`
`Eco RI
`
`PE38
`
`pULl3
`
`2 of 62
`
`BI Exhibit 1123
`
`

`

`U.S. Patent
`
`Mar. 30, 1999
`
`Sheet 2of17
`
`5,889,157
`
`PE40
`
`Ndel
`
`Hindm
`pULl1 (83(fv)-PE40)
`AmpR
`
`I MUTAGENESIS TO INTRODUCE THE C3-
`~ CONNECTOR BETWEEN 83!Fvl AND PE40
`
`GGPE GGS
`
`Ndel
`
`Hin m Soll
`pULl6 (83(fv)-Pf40/C3)
`
`AmpR
`
`PE38
`
`lsa11
`
`PE38
`
`NdeI
`
`Hindm Sall
`pULIT (83(fv)- PE38)
`
`AmpR
`
`FIG. 18.
`
`3 of 62
`
`BI Exhibit 1123
`
`

`

`U.S. Patent
`
`Mar. 30, 1999
`
`Sheet 3 of 17
`
`5,889,157
`
`N.de.I. J- ---Fv HEAVY CHAIN- -
`1 TTTAACTTTAAGAAGGAGATATACATATGGATGTGAAGCTGGTGGAGTCT
`S
`M D V K L V E
`SD
`S
`E V K L V E
`
`--------->
`51 GGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAAC 100
`G G G L V Q P G G S L K L S C A T
`G G G L V Q P G G S L
`
`50
`
`101 CTCTGGATTCACTTTCAGTGACTATTACATGTATTGGGTTCGCCAGACTC 150
`S G F T F S D Y Y M Y W V R Q T
`P
`
`151 CAGAGAAGAGGCTGGAGTGGGTCGCATACATTAGTAATGATGATAGTTCC 200
`S
`S N D D S
`I
`E K R L E W V A Y
`
`201 GCCGCTTATTCAGACACTGTAAAGGGCCGGTTCACCATCTCCAGAGACAA 250
`S R D N
`I
`A A Y S D T V K G R F T
`
`251 TGCCAGGAACACCCTCTACCTGCAAATGAGCCGTCTGAAGTCTGAGGACA 300
`A R N T L Y L Q M S R L K S E D T
`
`301 CAGCCATATATTCCTGTGCAAGAGGACTGGCCTGGGGAGCCTGGTTTGCT 350
`I Y S C A R G L A W G A W F A
`A
`BarnHI
`351 TACTGGGGCCAAGGGACTCTGGTCACTGTCTCCTCAGGCGGAGGCGGATC 400
`Y W G Q G T L V T V S S G G G G S
`<-- - - -Fv HEAVY CHAIN--J----------LINKER-
`
`-- -------LINKER---------------1 --Fv LIGHT CHAIN - --
`401 CGGTGGTGGCGGATCTGGAGGTGGCGGAAGCGATGTGCTGATGACCCAGT 450
`G G G G S G G G G S D V L M T Q S
`D V L M T Q S
`
`-----Fv LIGHT CHAIN---------->
`451 CTCCATTGAGTTTACCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGC 500
`S C
`I
`P L S L P V S L G D Q A S
`S L P V S L G
`? Q
`P L
`
`501 AGATCTAGTCAGATCATTGTACATAGTAATGGAAACACCTATTTAGAATG 550
`I V M S N G N T Y L E W
`S Q
`I
`R S
`
`551 GTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTT 600
`I Y K V S
`Y L Q K P G Q S P K L L
`
`FIG. 2A-1.
`
`4 of 62
`
`BI Exhibit 1123
`
`

`

`U.S. Patent
`
`Mar. 30, 1999
`
`Sheet 4of17
`
`5,889,157
`
`601 CCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGG 650
`N R F S G V P D R F S G S G S G
`
`651 ACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGT 700
`S R V E A E D L G V
`I
`T D F T L K
`
`701 TTATTACTGCTTTCAAGGTTCACATGTTCCATTCACGTTCGGCTCGGGGA 750
`Y Y C F Q G S N V P F T F G S G T
`
`HiodII I
`751 CAAAGCTGGAAATTAAAGCTTT ....... .
`I K A F -~ PE40
`K L E
`
`FIG. 2A-2.
`
`772
`
`721 CACATGTTCCATTCACGTTCGGCTCGGGGACAAAGCTGGAAATTAAATAA 770
`*
`I K
`H V P F T F G S G T K L E
`
`EcoRI
`771 TGAATTCC . .
`* - :iio-
`TERM
`
`FIG. 28.
`
`779
`
`5 of 62
`
`BI Exhibit 1123
`
`

`

`U.S. Patent
`
`Mar. 30, 1999
`
`Sheet 5 of 17
`
`5,889,157
`
`J
`
`10
`I
`ng/nll TOXIN
`.JA.
`FIG.
`
`100
`
`1000
`
`IOOOO
`
`0
`
`0
`
`~IOo--~~~~~~~~~~~~~~~~~~-,
`a::
`~ 90
`~ 80
`~ 10
`~ 60
`Q.. 8 50
`~
`:E 40
`::::>
`~ 30 _._ 83(fv)PE38K
`-0- 83(fv)PE38K+ HB21
`20
`:
`IO 4- 83(fv)PE38l ... 83
`:
`
`0
`.01
`
`.I
`
`I
`ng/111 TOXIN
`
`FIG.
`
`.JS.
`
`10
`
`100
`
`6 of 62
`
`BI Exhibit 1123
`
`

`

`U.S. Patent
`
`Mar. 30, 1999
`
`Sheet 6of17
`
`5,889,157
`
`3000.....---------- - - - - --
`
`2500
`
`2000
`
`z
`~ 1500
`
`IOOO
`
`500
`
`-<r B3(Fv)PE38
`-o- Bl (Fv) PE38
`-tr B5(fv)PE38
`
`IO
`
`20
`30
`IMMUNOTOXIN
`ng
`FIG. 4A.
`
`40
`
`50
`
`=> ';le
`~ 40
`,...,
`:c
`
`20
`o-l-.-,~~~....::~~~~...,...,.....,~
`.01
`.I
`10
`100
`1000
`10000
`ng/ml
`
`-c>-- B3(Fv)PE38
`-o- Bl(fv)PE38
`
`--6- 85(Fv) PE38
`
`FIG. 48.
`
`7 of 62
`
`BI Exhibit 1123
`
`

`

`U.S. Patent
`
`Mar. 30, 1999
`
`Sheet 7of17
`
`5,889,157
`
`120...---- - - -- - - -- - - - - - - - - - .
`
`100
`c
`:z::
`=>
`~ BO
`Cft 60
`
`(.!)
`
`N
`
`40
`
`'°-
`~ 20
`
`0-t---r-...-.-r-rrr-rr-----r--r-..-...-Tnrrr----r--,.-r..,...,.,.,r-r-r<-~---,~.......i
`.01
`.I
`I
`10
`IOO
`JtM COMPETITOR
`
`-0- 83{fv)PE38
`--0-8HFv)PE38
`FIG. 5A.
`
`-er 85(fV)PE38
`
`120~----------------.
`
`IOO
`
`c
`~ 80
`
`0
`a::i
`
`60
`
`C.!>
`
`°'
`a;
`
`40 -"">
`
`~ 20
`o--~~~~~~--~~~...........-........------...................
`10
`100
`.I
`I
`.01
`JlM COMPETITOR
`
`-0- B3ffv)PE38
`-o- BHfv)PE38
`FIG. 58.
`
`-fr- 85 (FY) PE38
`
`8 of 62
`
`BI Exhibit 1123
`
`

`

`U.S. Patent
`
`Mar. 30, 1999
`
`Sheet 8of17
`
`5,889,157
`
`9 of 62
`
`BI Exhibit 1123
`
`

`

`U.S. Patent
`
`Mar. 30, 1999
`
`Sheet 9of17
`
`5,889,157
`
`10
`
`6
`5
`_. 4
`14.1
`0
`3
`:a.c
`ao
`W')
`14.1 a.. 2
`
`I ->
`...... -W')
`e -~ :»
`
`c:g
`
`0
`0
`
`10
`
`20
`
`0.8
`
`-
`<.:> -
`
`~ 0.6
`
`14.1
`
`~ 0.4
`a::
`~ 0.2
`::>
`I -
`
`70
`
`80
`
`90
`
`60
`50
`40
`30
`MIN AFTER INJECTION
`FIG. 7.
`0.8 -
`
`W')
`
`~ 0.6
`,....,
`"""
`u; 0.4
`a:
`0
`!i 0.2
`
`I -
`
`0.0
`
`0 2 4 6 8
`12
`10
`FIG. BA. TIME<DAYS)
`
`W')
`
`0.8
`
`-
`z
`~ 0.6
`,....,
`"""
`Cl) 0.4
`a::
`0
`z e 0.2
`0.0
`10
`0 2 4 6 8
`TIME <DAYS)
`FIG. BC.
`
`ID
`2 4
`Fl& 88. TIME CDAYS)
`
`0.8
`
`W')
`
`-
`-
`~ 0.6
`""" ~ 0.4
`a:
`0
`~ 0.2
`
`I -
`
`12
`
`0.0
`0 2 4 6 8
`10
`TINE <DAYS)
`FIG. 80.
`
`12
`
`10 of 62
`
`BI Exhibit 1123
`
`

`

`U.S. Patent
`
`Mar. 30, 1999
`
`Sheet 10 of 17
`
`5,889,157
`
`-BSHFrt
`CH1
`VH
`-B5HFr4
`
`pCR·85VH
`
`pCR·BSVL
`
`PCR AMPLIFY VARIABLE
`SEGMENTS, ANNEAL PCR
`PRODUCTS SEPARATELY OR
`COMBINED TO THE TEMPLATE
`~
`~~
`..-----. l - - - .....----......
`;1 a3vff H B3VL H ma ~
`p~l7 ss-DNA
`~
`)
`I EXTEND ANO LIGATE
`t TRANSFRON E.cDli OH~
`
`L
`
`83VH
`
`B5VL
`
`PE38
`
`p83-5<fv)PE38
`
`OR
`
`L
`
`85VH
`
`83VL
`
`PE38
`
`p85· 3{fv)PE38
`
`OR
`
`L c 85\'tt H 85VL H PE38 )
`
`p85Cfv)PE38
`
`FIG. 9.
`
`11 of 62
`
`BI Exhibit 1123
`
`

`

`U.S. Patent
`US. Patent
`
`Mar. 30, 1999
`Mar. 30, 1999
`
`Sheet 11 0f17
`Sheet 11 of 17
`
`5,889,157
`5,889,157
`
`|20
`
`astrmaa
`
`
`
`
`.l
`
`I
`
`I!)
`
`ma
`
`IOOOngImi
`
`B3VH-BSVL-PE38
`
`+1=0HOUR5
`+IHOUR31I§BPBSAT
`+2 nouns I" was
`AT 37%;
`
`+ f= o nouns
`_._ | Hog}? PBS AT
`mm;
`
`+ 2 nouns m PBS
`
`100
`
`g
`r;
`4 ...1
`
`ii an
`§ 3 so
`; “5
`40
`3 :2
`
`"°
`
`FI6‘. IOA.
`
`20
`
`0
`.0:
`I20
`
`IOU
`
`g
`'5 a:
`E E "0
`g E 50
`E 3‘ 40
`
`m
`
`20
`
`FIG I08. o
`
`I00
`
`so
`
`5
`3 _|
`2% so
`g g
`3 ‘5 40
`3 :1
`.55
`
`20
`
`FIG. I06: 0
`
`
`
`.0!
`
`.1
`
`l
`
`10
`
`[00
`
`I000 ngIml
`
`
`
`+t=0HOURS
`_,_ I mum; PBS AT
`+2HOURS m PBS
`mm
`
`.01
`
`.t
`
`1
`
`I0
`
`:00
`
`1000 ng/rnl
`
`12 of 62
`
`BI Exhibit 1123
`
`12 of 62
`
`BI Exhibit 1123
`
`

`

`'I
`Ul
`~
`~ -.
`oc
`-. oc
`Ul
`
`....,1
`~
`~
`0
`N
`~
`~
`~
`00 :r
`
`\0
`~
`
`~
`
`.,.Q
`(.-l
`
`~
`3:
`
`(D = ""'"
`
`""'"
`~
`""d
`•
`rJ'1
`•
`Cj
`
`FIG. 11 B.
`
`HumB3Vl GVPDRFSGSGSGTDFRLKISRVEAEDVGVYYC FQFSHVPFT FGQGTKVEIK
`GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC MQGLQTPQT FGQGTKVEIK
`GM607
`GVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC FQFSHVPFT FGSGTKLEIK
`B3VL
`
`I
`100 104
`
`I
`
`CDR3
`
`I
`83
`
`*
`
`**
`
`HumB3Vl DVLMTQSPLSLPVTPGEPASISC RSSQIIVHSNGNTYLE WYLQKPGQSPQLLIY KVSNRFS
`DIVMTQSPLSLPVTPGEPASISC RSSQSLLHSNGYNYLD WYLQKPQQSPQLLIY LGSNRAS
`GM607
`DVLMTQSPLSLPVSLGDQASISC RSSQIIVHSNGNTYLE WYLQKPGQSPKLLIY KVSNRFS
`B3VL
`
`CDR2
`
`I
`
`45
`
`I
`
`CDRl
`
`14 15 17 18
`
`1 ( ((
`
`I I
`
`FIG. 11A.
`
`* *
`
`HumB3VH RFTISRDNSKNTLYLQMNSLRAEDTAIYSCAR TLAWTAWRAY WGQGTLVTVSS
`56Pl'CL RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR RSARTYYFDY WGQGTLVTVSS
`RFTISTDNARNTLYLQMSRLKSEDRAIYSCAR GLAWGAWFAY WGQGTLVTVSS
`B3VH
`
`CDR3
`
`I
`
`l I (r I
`
`74 7(5 82a 82b 83 84
`
`l
`
`HumB3VH DVKLVESGGGVVQPGRSLKLSCATSGFTFS DYYMY WVRQAPGKGLEWVA YISNDDSSAAYSDRVKG
`56Pl'CL QVELVESGGGVVQPGRSLRLSCAASGFTFS SYAMH WVRQAPGKGLEWVA VISYDGSNKYYADSVKG
`DVKLVESGGGLVQPGGSLKLSCATSGFTFS DYYMY WVRQTPEKRLEWVA YISNDDSSAAYSDTVKG
`B3VH
`I
`
`CDR2
`
`I ( (
`40 42 44
`
`CDRl
`
`I
`
`I
`
`I
`16
`
`I
`11
`
`* *
`
`I
`
`*
`
`*
`
`13 of 62
`
`BI Exhibit 1123
`
`

`

`U.S. Patent
`
`Mar. 30, 1999
`
`Sheet 13 of 17
`
`5,889,157
`
`FIG. 12A.
`
`FIG. 128.
`
`FIG. 12C.
`
`FIG. 120.
`
`PstI
`I
`
`Hindfil
`I
`
`EcoRI
`I
`
`PE38
`
`MUTAGENESIS
`OUGO'S 1-4
`t
`PE38
`
`p83HUNVH -VL-PE38
`
`MUTA GENESIS
`OLIGO'S 5-8
`
`l
`
`HUMVL
`
`PE38
`
`p83Vw HUMVL -PE38
`
`MUTAGENESIS
`OUGO'S 1-4
`
`•
`
`HUNVH
`
`HUNVL
`
`PE38
`
`p83HUNV}rHUNVL PE38
`
`1 00 ----....--~------.
`
`0 83( Fv) PE38
`
`80
`
`60
`
`40
`
`20
`
`~ B3HUNvH -HUMvL -PE38
`
`~ HUMB3(fv)PE38
`
`0-'---~-~~~--------t
`
`FIG. 14.
`
`14 of 62
`
`BI Exhibit 1123
`
`

`

`U.S. Patent
`
`Mar. 30, 1999
`
`Sheet 14 of 17
`
`5,889,157
`
`1soo~------------------..
`
`1200
`
`800
`
`400------..--r---.--,------.,.-~-~-~~---'
`50
`JO
`20
`30
`0
`40
`FIG. l.JA.
`ng INNUNOTOXIN
`
`JOO
`
`:c
`H')
`
`Q+._-,.,...........,--.-.-.........., ........................ ....-.,...,..........,P::;::;:~l)oo.~~--..........j
`.OI
`.001
`.I
`I
`10
`100
`IOOO
`10000
`F/6. /SB.
`
`ng/ml
`
`120....-----------------~
`~ 100
`0
`co 80
`<.o
`~ 60
`co
`-
`40
`in
`~ 20
`Vt
`O+-....--. ............. .........--.--.-.-.,..,..,..,,...--....-.-----.---...-.....,...,........,..,r---..-.--........,..,r-n1
`l:!"J~. /.' "'C. 10-3
`I01
`I0-2
`IO-I
`100
`102
`rt~. ._,
`[COMPETITORJ
`J1N
`-<r- 83(f v) PE38
`---&- 83HUMVH-VL -PE38
`
`--()-- B3VH-HUMVL-Pf38
`•
`B3HUMVw HUMVL -PE38
`•
`HUNB3(fv)PE38
`
`15 of 62
`
`BI Exhibit 1123
`
`

`

`U.S. Patent
`
`Mar. 30, 1999
`
`Sheet 15 of 17
`
`5,889,157
`
`GAGGTGCAGCTGGTGGAATCTGGAGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAA
`E V Q L V E S G G G L V K P G G S L K
`E V Q L V E
`S G G G L V K P G G S
`L
`CTCTCCTGTGCAGCCTCTGGATTCATTTTCAGTGACAATTACATGTATTGGGTTCGC
`I F S D N Y M Y W V R
`L S C A A S G F
`CDR 1
`CAGACTCCGGAGAAGAGGCTGGAGTGGGTCGCAACCATTAGTGATGGTGGCACTTAT
`Q T P E K R L E W V A T
`I
`S D G G T Y
`COR 2
`ATCGACTATTCAGACAGTGTGAAGGGGCGATTCACCATCTCCAGAGACAATGCCAAG
`I D Y S D S V K G R F T
`I
`S R D N A K
`
`AATAATCTGTACTTGCAAATGAGCAGTCTGAGGTCTGAGGACACAGGCATGTATTAT
`N N L Y 1 Q M S S L R S E D T G M Y Y
`
`TGTGGAAGGAGTCCGATCTACTATGATTACGCCCCGTTTACTTACTGGGGCCAAGGG
`Y Y D Y A P
`F T Y W G Q G
`C G R S P
`I
`CDR 3
`ACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCC
`C L V T V S A A K ~ ~ P
`P S V Y P L A
`
`C·'.::TGGATCTGCT
`P
`6
`S A
`
`FIG. 15A.
`
`GATGTTGTGATGACCCAGACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCC
`D V V M T Q T P L S L P V S L G D Q A
`D V V M T Q T P L
`S
`L P V
`S
`L G D
`TCCATCTCTTGCAGATCTAGTCAAAACCTTGTACACAGTGATGGAAAAACCTATTTA
`S C R S S 0 N L V H S P G K T Y L
`S
`I
`CDR 1
`CATTGGTTCCTGCAGAAGCCTGGCCAGTCTCCAACGCTCCTGATCTACAAAGTTTCC
`Y K V S
`_H W F L Q K P G Q S P T L L
`I
`CDR 2
`AACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTC
`N R F S G V P D R F S G S G S G T D F
`
`ATACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAA
`I
`L K
`I
`S R V E A E D L G V Y F C S
`0
`
`AGTACACATGTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGGGCT
`S T H V P L T F G A G T K L E L K R A
`CDR 3
`GATGCTGCACCAACTGTATCCATCTTCCCACCA
`D A A P ~ V S
`I
`F P P
`FIG. 158.
`
`16 of 62
`
`BI Exhibit 1123
`
`

`

`U.S. Patent
`
`Mar. 30, 1999
`
`Sheet 16 of 17
`
`5,889,157
`
`GAGGTGAAGCTGGTGGAATCTGGAGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAAA
`E V K L V E S G G G L V Q P G G S L K
`E V K L V E S G G G L V Q P G
`CTCTCCTGTGCAACCTCTGGATTTACTTTCAGTGACTATTACATGTATTGGGTTCGC
`L S C A T S G F T F S D Y Y M Y W V R
`CDR 1
`CAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCATACATTAGTAATGGTGGTGGTAGC
`S N G G G S
`I
`Q T P E K R L E W V A Y
`CDR 2
`ACCTATTATCCAGACACTGTAAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAG
`I D R D N A K
`T Y Y P D T V K G R F T
`
`AACACCCTGTACCTGCAGATGAGCCGTCTGAAGTCTGAGGACACAGCCATGTATTAC
`N T L Y L Q M S R L K S E D T A M Y Y
`
`TGTGCAAGGGGGCTCTCTGATGGTTCCTGGTTTGCTTACTGGGGCCAAGGGACTCTG
`C A R G L S D G S W F A Y W G Q G T L
`CDR 3
`GTCACTGTCTCCTCAGGCGGAGGCGGATCCGGT
`V T V S S G G G G S G
`FIG. 16A.
`
`GATGTTTTGTTGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCC
`D V L L T Q T P L S L P V S L G D Q A
`L P V S L
`D V L L T Q T P L S
`TCTATTTCTTGTAGATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTA
`I V H S N G N T Y L
`S
`S C R S S 0
`I
`S
`CDR 1
`GAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCC
`~ W Y L Q K P G Q S P K L L
`I Y K V S
`CDR 2
`AACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTC
`N R F S G V P D R F S G S G S G T D F
`
`ACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAA
`0
`S R V E A E D L G V Y Y C F
`I
`T L K
`
`GGTTCACATGTTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAGCGGGCT
`I K R A
`G S H V P F T F G S G T K L E
`CDR 3
`GATGCTGCACCAACTGTATCCATCTTCCCACCA
`I F P P
`6
`D A A P ~ V
`FIG. 168.
`
`17 of 62
`
`BI Exhibit 1123
`
`

`

`00 '° ... ,,.....
`00
`...
`VI
`
`-.....l
`VI
`
`--l
`......
`0 ....,
`--l
`......
`~ ...
`'JJ =(cid:173)~
`
`~
`......
`9
`~
`
`~
`
`~ = :-i
`
`~ = .....
`~ .....
`~
`•
`'J:J
`~ •
`
`FIG. 18.
`
`CGTTCGGCCAGGGTACCAAGGTCGAAATTAAA
`ACACTCAAGATCAGCAGAGTGGAGGCTGAGGACGTCGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCATTCA
`GCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTC
`GTCAGATCATTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCACAGCT
`GATGTGCTGATGACCCAGTCTCCATTGAGTTTACCTGTCACCCCGGGAGAGCCGGCCTCCATCTCTTGCAGATCTA
`VL:
`
`LINKER: ggcggaggcggatccggtggtggcggatctggaggtggcggaagc
`
`GGGGAGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCCTCA
`AACACCCTCTACCTGCAAATGAACCGTCTGCGCGCCGAGGACACAGCCATATATTCCTGTGCAAGAGGACTGGCCT
`TAGTAATGATGATAGTTCCGCCGCTTATTCAGACACTGTAAAGGGCCGGTTCACCATCTCTAGAGACAATAGCAAG
`GATTCACTTTCAGTGACTATTACATGTATTGGGTTCGCCAGGCCCCGGGCAAGGGCCTGGAGTGGGTCGCATACAT
`GATGTGAAGCTGGTGGAGTCTGGGGGAGGCGTCGTGCAGCCCGGGCGCTCCCTGAAACTCTCCTGTGCAACCTCTG
`VH:
`
`(KVSNRFS)
`
`(RSSQIIVHSNGNTYLE) WYLQKPGQSPKLLIY
`
`GVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC
`DVLMTQSPLSLPVSLGDQASISC
`VL:
`
`FIG. 17.
`
`(FQGSHVPFT) FGSGTKLEIK
`
`(DYYMY) WVRQTPEKRLEWVA (YISNDDSSAAYSDTVKG)
`
`(GLAWGAWFAY) WGQGTLVTVSS
`
`RFTISRDNARNTLYLQMSRLKSEDTAIYSCAR
`DVKLVESGGGLVQPGGSLKLSCATSGFTFS
`VH:
`
`GGGGSGGGGSGGGGS
`LINKER:
`
`18 of 62
`
`BI Exhibit 1123
`
`

`

`5,889,157
`
`1
`HUMANI ZED 83 ANTrBODY FRAGMENTS,
`FUSION PROTEINS, ANO USES THEREOF
`
`Tbc preseot applicatioo is a continuation-in-part of U.S.
`patent application Ser. No. 07n 67,331, filed on Sep. 30,
`1991, oow abandoned, which is a continuation-in-part of
`U.S . patent application Ser. No. 07/596,289 filed on Oct. 12,
`1990 issued as U.S. Pat. No. 5,242,813 both of which are
`hereby incorporated by reference.
`
`2
`has been s hown that certain single chain antigen binding
`proteins made from the Fv portions of the heavy and light
`cbain of antibodies beld together by a polypeptide linker can
`have the same binding properties as their fuo length two
`s cbain counterparts (Bird ct al., Science, 242: 423-26 (1988)
`and Huston et al., Proc. Natl. Acad. Sci. USA, 85: 5879-83
`(1988)). 11 bas also been shown that, in some cases, fusion
`proteins composed of single chain antibodies linked to
`toxins may retain the binding capacity of the single chain
`10 antibody as well as the activity of the toxin (Chaudhary et
`al., Nature, 339: 394-97 (1989); Batra e t al., J. Biol. Chem.,
`265: 15198-15202 (1990); Batra et al., Proc. Natl. Acad.
`Sci. USA 86: 8545-8549 (1989); Cbaudhary et al., Proc.
`Natl. Acad. Sci. USA 87: 1066-1070 (1990)).
`Receptor proteins have often been used as immuootoxin
`targets because they are cell surface proteins which are often
`overexpressed in various cancers (Brinkmann and Pastao,
`Biocltem. Biophys. Acta., 1198: 27-45 (1994)) and thus
`provide cancer-specific targets. For example, single chain
`immunotoxins have been made consisting of the Fv domain
`of ao antibody directed al the interleukin 2 receptor
`(Chaudbary ct al., Nawre, 339: 394-97 (1989) aod Batra ct
`al., J. Biol. Chem. 265: 15198-15202 (1990)) or at the
`transferrin receptor (Batra et al., Proc. Natl. A cad. Sci. USA
`86: 8545-49 (1989)) fused to truncated forms of PE or
`diphtheria toxin (Chaudhary et al., Proc. Natl. Acad. Sci.
`USA 87: 9491-94 (1990)). Although receptor proteins are
`overcxpressed on many cancers, tbey may still be present on
`healthy cells and therefore often do not provide the defined
`caocer specificity desired for an immu notoxio.
`Since the number of antibodies Lbat react preferentially
`with carcinomas is limited, the identification and character(cid:173)
`ization of addilioaal "cancer specific" antibodies that would
`react with aU or most of the cells in a tumo r and with
`relatively few normal cells and tissues is desirable. lo
`addition, recombinant immuaotoxias are known to degrade
`over time both in vitro and in vivo. It would be desirable to
`obtain immunotoxins tbat show a redticed rate <>f degrada-
`tion and therefore require less frequent administration.
`Finally, with repeated use, muri ne antibodies and fusion
`proteins containing muriae antibodies, like any other foreign
`protein, may ultimately prove immuoogenic and invoke an
`immune response in the treated organism. It would be
`desirable to produce targeting moieties and iromunotoxios
`having reduced antigenic potential. As will be explained
`herein, these advantages and others are provided by the
`present invention.
`
`30
`
`BACKGROUND OF THE INVENTION
`The subject invention relates to tumor-specific recombi(cid:173)
`nant antibody fragments, to molecules incorporating such
`fragments such as immuootoxins and to uses thereof. Exem(cid:173)
`plary embodiments of the invention ioclude immuootoxins 15
`comprising Pseudomooas exotoxins fused to the Fv regions
`of monoclonal antibodies Bl, B3, and BS which have tumor
`specificity and which may be used in the treatment of
`mammalian cancer.
`Monoclonal antibodies Bl, B3, and BS are recently 20
`isolated murioe antibodies directed against a carbohydrate
`
`antigen in the Lewis1' (Le1
`') family (Pastan et al. Cancer
`Res., 51: 3781-3787 (1991)). The Le-'" antigens are found on
`lhe surface of many mucinous carcinomas of the colon,
`stomach, ovaries, breast, Jung as well as some epidermal 25
`carcinomas. Because they react with only a limited number
`of normal tissues, these antibodies are ideal candidates for
`use in the treatment and diagnosis of cancer.
`lo order to create a cytotoxic agent that specifically
`aliacks cancer cells, an antibody or its fragments may be
`used as the targeting moiety of an irorouootoxio. lo such
`immuootoxins, the targeting moiety typically replaces tbe
`cell binding domain of a cytotoxio molecule (e.g. domain 1
`of Pseudomooas exotoxin (PE) or the B chain of Diphtheria
`toxin) and acts to specifically direct the cytotoxio to its target
`cell (as determined by the specificity of Lbe targeting
`moiety). As a result, only cells wbicb are recognized by tbe
`targeting moiety are efficiently killed and cells which are not
`recognized are spared (for a review see Brinkmann and 40
`Pastan, Biochem. Biophys. Ac10., 1198: 27-45 (1994)).
`lrnmunotoxias were first made by chemically coupling
`anl ibodies to cytotoxic molecules. Thus, for example, mono(cid:173)
`clonal antibody B3 bas been chemically coupled to at least
`two different forms of Pseudomoaas exotoxia (PE) (U.S. 45
`Pat. No. 4,545,985). One of Lbese is the full length toxin (PE)
`and the other a truncated derivative (PE40) (Kondo et al., .!.
`Biol. Chem., 263: 9470-75 (1988) and Pai et al., supra).
`Both of these immunotoxins have been shown to be selec(cid:173)
`tively cytotoxic to tumor cells that contain the B3 antigen on 50
`their surface, aod tbese imrnuooloxios have been sbown to
`cause complete tumor regression in mice bearing human
`tumor xcoografts (Pai ct al., Proc. Natl. 1\cad. Sci. USA, 88:
`3358-62 (1991)).
`Although chemically coupled immunotoxias are useful
`they have several undesirable properties. For example, the
`chemical modifications can change the antibody and affect
`its binding LO the antigen. Funhem1ore, the purified immu(cid:173)
`notoxins are a heterogeneous mixture of antibody-toxin
`mo lecules connected Lo each other v ia different positions on
`tbe antibody and the toxin. Thus, Pseudomooas exotoxio, for
`example, can be coupled either 10 the light- or heavy-chain
`of the antibody and to different positions on each of these
`chains.
`To overcome the limitations of chemically conjugated 65
`immuaotoxias, chimeric immuaotoxios have been made as
`recombinant, single chain, antibody-toxin fusion proteins. It
`
`35
`
`SUMMARY OF TI-IE INVENTION
`
`The present invention provides for recombinant single
`chain antibodies and fusion proteins, such as iromunotoxins,
`employing these ant ibodfos. ln particular, this invention
`provides for recombioaotly produced humanized single-
`55 chain antibodies comprising humanized variable light and
`heavy (Fv) regions of antibodies that have the binding
`speci fi city of monoclonal antibody BJ, B3 or BS. These
`antibodies provide carcinoma-specific targeting moieties
`suitable for use in cytotoxic fusion proicins. Particularly
`60 preferred are humanized single-chain Fv regions of Bl, B3
`or BS.
`la one embodiment, the single-chain antibody is a human(cid:173)
`ized B3(Fv). Particularly preferred is an antibody compris(cid:173)
`ing a humanized variable heavy chain, more specifically a
`humanized variable heavy chain having the amino acid
`sequence designated HumB3V11 in FIG. 11. Another pre(cid:173)
`ferred variant is an antibody comprising a humanized vari-
`
`19 of 62
`
`BI Exhibit 1123
`
`

`

`5,889,157
`
`3
`able lighl chain, more specifically a humanized variable
`lighl chain baving tbe amino acid sequence designated
`HumB3V 1.. in FTG. ll. Ye1 anolber preferred humanized
`antibody is one comprising both a bumaoizecl variable ligb1
`chain and a bumaoized variable heavy chain. Particularly 5
`preferred is an antibody comprising a bumaoizecl variable
`heavy cbain baviog the amino acid sequence designated
`HumB3V17in FIG. 11 and a humanized variable ligh1 cha.in
`baviog 1be amino acid sequence designated HumB3 V 1.. in
`FIG. 11. Still yet another preferred humanized antibody is 10
`one comprising a humanized variable beavy ebain having
`the amino acid sequence designated HumB3V11 in FIG. 11
`and a humanized variable ligbt cbain baving lbe amino acid
`sequence designated HumB3VH in FIG. ll in which the
`serine at the position designated as 82b in FIG. ll, is 15
`replaced with arginine.
`lo any of the single chain antibodies described above lhe
`variable heavy region and the variable light region may be
`joined by a linker. One par1icularly preferred linker is
`(Gly4Ser)3 (SEQ ID N0:32).
`This invention also provides for single-chain fosion pro(cid:173)
`teins incorporating any of tbe above-described single-cbaio
`antibodies. The fusion proteins comprise tbe single chain
`antibodies recombinantly fused to an effector molecule. The
`effector molecule may be a cy101oxio such as Pseudomooas
`exotoxin and more preferably is either PE38, P40,
`PE38KDEL, or PE38REDL. Thus preferred fusion proteins
`include HUMB3(Fv)-PE38, HUMB3(Fv)-PE40, HUMB3
`(Fv)-PE38KDEL and HUMB3(Pv)-PE38REDL.
`TI1e fusion proteins may also include a linker belween the
`variable heavy (VH) and the variable lighl (V L) chain regions
`of tbe Fv fragmen t. One preferred linker is the peptide linker
`(Gly4 Serh (SEQ ID N0:32). "fbe fusion proteins may also
`include a connector between the Fv region and the effector 35
`molecule. A particular preferred connector is SGGPEGGS
`(SEQ ID N0:44).
`1bis invention also provides for a method of detecting the
`presence or absence of a cell bearing a LewisY carbohydrate
`antigen in a patient. The method involves removing a tissue
`or fluid sample from the pa1ien1; adding any of the above(cid:173)
`dcscribcd antibodies to the sam1>le aod dctectiog for the
`presence or absence of a binding complex between ihe
`antibody and the antigen.
`lo another embodiment, this invention provides for a 45
`method of killing or inhibiting the growth of cells bearing a
`Lewis1
`' antigen in a patient. This method includes adminis(cid:173)
`tering to the patient a pharmaceutical composition in an
`amount sufficient to kill or inhibit the growth of 1be cells.
`The pharmaceutical composition wiU include any of tbe so
`above-described antibodies or fusion proteins.
`
`40
`
`4
`FIG. lb shows lhe coostruction LMB7, the immunoloxin
`B3(Fv)-PE38 with a "C3 connector" between the Fv region
`and the PE38 cytotoxio (GGGGSGGGGSGGGGS linker=
`SEQ lD N0:32; SGGPEGGS C3 conoector~SEQ ID
`N0:44).
`FIG. 2 shows the nucleotide sequences encoding the
`heavy and light chain Fv region of monoclonal antibody B3
`(SEQ ID N0:33). (a) The heavy chain Fv coding region
`extends from position 30 to 383, the Jigh1 chain Fv gene
`from position 433 10 767 and the linker from 384 10 432. The
`deduced amino acid sequence is sbowo in plain letters (SEQ
`ID N0:34); below in italic letters is nhe protein sequence
`detcrmiocd by Edman scqucnciog of tbe ani-ibody (SEQ ID
`N0:56). The first amino acid encoded by the cloned heavy
`chain Fv gene is Asp instead of Glu due to the oligooucle(cid:173)
`otide primer used al position 456-465. This is 1be region
`where 1he PCR cloning artifact was repaired. This sequence
`encodes 1he same amino acids as the original B3 ligh1 chain
`gene bu1 uses other codons. Homology comparisons to the
`20 known nucleotide sequence of PACT Jg kappa chain (Taub
`cl al., J. Biol. Chem., 264: 59-65 (1989)) wbjcb is most
`homologous lo the B3 light chain iodica1es that the original
`sequence was most probably CTCrCCCTG (SEQ ID
`N0:37) instead ofTTGAGTITA(SEQ ID N0:38). 1nus the
`25 natural B3 light chain gene bas a sequence repetition
`5-(CCAGTCT[CC)ACTCTCC]-3' (SEQ ID N0:39)
`between positions 445 and 465 which is responsible for the
`incorrect primer annealing in PCR. (b) Sequence al 1he
`3'-end of the light chain for expression of the single chain
`JO B3(Fv) alone (SEQ fD N0:35 and amino acid sequence
`SEQ ID N0:36). (SD)- Shine Dalgarno consensus
`sequence; (*)-translation stop signal. (Term) transcription
`terminator.
`FIG. 3(n) represents lhe toxicity of B3(Fv)-PE38KDEL
`on different cell lines. Cytotoxicity assays were performed
`as described in Example 7. (b): Inhibition of the cy101oxicity
`of B3(Fv)-PE38KDEL by monoclonal amibody B3. Com(cid:173)
`petition by monoclonal antibody B3 was performed on A431
`cells as described in Example 7.
`FIG. 4 shows the ADP-ribosylalion and cytotoxic ac1ivi-
`1ies of Bl(Fv)-PE38, B3(Fv)-PE38, BS(Fv)-PE38 recombi(cid:173)
`nant immunotoxins. (A) A DP-ribosylalion activity was
`determined by lhe incorporation on 1 4C-NAD into acid(cid:173)
`prccipitable material using elongation factor 2 enriched
`wheat-germ extract (Collier and Kandel, 1971). (B) Cyto-
`toxic i1y towards A431 cells was measUcred by the inhibition
`of incorporation of 3H-leucine into cell protein, following 2
`hours (open symbols) or 20 hours (solid symbols) of incu-
`bation of the cells with serial dilutions of immuno1oxins in
`PBS+0.2% BSA.
`FIG. S shows antigen binding of Bl(Fv)-PE38, B3(Fv)(cid:173)
`PE38 and B5(Fv)-PE38. Antigen binding was estimated by
`competition of. [1251]-Bl lgG (A) or [ 125l]-B3 lgG (B)
`55 binding to A431 cells at 4° C.
`FIG. 6 shows stability data for Bl(Fv)-PE38, B3(Fv)(cid:173)
`PE38, and B5(Fv)-PE38. Tbe immuno1oxins were diluted in
`PBS to 0.1 mg/ml and incubated at 37" C. for 4 hours. {A)
`The molecular fo rms of lhc immunotoxins were lban ana-
`60 lyzed by size exclusion chromatography al 4° C. The mono(cid:173)
`mer peak elutes at 18-20 ml, wbile the aggregates elute at
`11-13 ml. Chromatograms of the proteins prior to incuba(cid:173)
`tion at 37° C. are shown by broken Lines. The proteins after
`the incubation at 37° C. are shown by solid lines. (B)
`65 Cytotoxic activity of immunotoxins before (open symbols)
`or after (solid symbols) incubation al 37° C. 0 1ber details are
`as in FIG. S(B).
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`FIG. l n illus1ra1es the strategy for the cloning of the heavy
`and light chain Fv genes of monoclonal antibody B3 and the
`coos1ructioo of expression vectors (e.g., plasmids) for the
`expression of B3(Fv) immunotoxins. The cloning strategy is
`a variation of thal previously described (Cbaudhary ct al.,
`Proc. Natl. Acad. Sci. USA, 87: 1066-70 (1990)). The
`plasmid pVC38H, which is used as a vector for construction
`of immuootoxins fr