United States Patent
`
`r19J
`
`Queen et al.
`
`[54) HUMANIZED IMMUNOGLOBULINS
`
`[75)
`
`Inventors: Cary L. Queen, Los Altos; Harold E.
`
`Selick, Belmont, both of Calif.
`[73] Assignee: Protein Design Labs, Inc., Mountain
`
`View, Calif.
`
`[21) Appl. No.: 634,278
`Dec. 19, 1990
`
`[22) Filed:
`
`Related U.S. Application Data
`
`I lllll llllllll Ill lllll lllll lllll lllll lllll 111111111111111111111111111111111
`
`US005530101A
`[111 Patent Number:
`[45) Date of Patent:
`
`5,530,101
`Jun. 25, 1996
`
`Neuberger et al., "A hapten-specific chimeric IgE antibody
`with human physiological effector function," Nature
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`Monison, S. L., "Transfectomas Provide Novel Chimeric
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`Sahagan ct al., "A Genetically Engineered Murine/Human
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`Liu et al., "Expression of mouse::human immunoglobulin
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`Better et al., "Escherichia coli Secretion of an Active
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`[52) U.S. CI . ..................................... 530/387.3; 530/387.1;
`(51) Int. Cl.6 ......................... A61K 39/395; C07K 16/28
`[58) Field of Search ................................ 424/85.8, 133.l,
`
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`
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`
`ABSTRACT
`
`Primary Examiner-Lila Feisee
`Allomey, Agent, or Fim1-Townsend and Townsend and
`Crew
`(57]
`Novel methods for producing, and compositions of, human­
`ized immunoglobulins having one or more complementarity
`determining regions (CDR's) and possible additional amino
`acids from a donor irnrnunoglobulin and a framework region
`from an accepting human irnmunoglobulin are provided.
`Each humanized immunoglobulin chain will usually com­
`prise, in addition to the CDR's, amino acids from the donor
`immunoglobulin framework that are, e.g., capable of inter­
`acting with the CDR's to effect binding affinity, such as one
`
`or more amino acids which are immediately adjacent to a
`chains may each be designed by using any one or all of
`
`CDR in the donor immunoglobulin or those within about 3
`A as predicted by molecular modeling. The heavy and light
`
`various position criteria. When combin.ed into an intact
`antibody, the humanized irnmunoglobulins of the present
`invention will be substantially non-immunogenic in humans
`and retain substantially the same affinity as the donor
`
`immunoglobulin to the antigen, such as a protein or other
`
`compound containing an epitope.
`
`13 Claims, 55 Drawing Sheets
`
`1 of 139
`
`BI Exhibit 1136
`
`

`

`5,530,101
`Page 2
`
`in
`
`OTHER PUBLICATIONS
`
`Reichmann et al., "Reshaping human antibodies for
`
`therapy", Nature 332:323-327 (1988).
`Bird et al., "Single-chain Antigen-Binding Proteins", Sci­
`ence 242:423-426 (1988).
`Huston et al., "Protein engineering of antibody binding sites:
`Recovery of specific activity in an anti-digoxin single-chain
`Fv analogue produced in Escherichia coif', Proc. Natl.
`
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`
`York (1986) p. 283.
`Uchiyama et al. Journal of Immunology vol. 126 No. 4 1981,
`
`Kirkman et al. Journal of Expt. Med. vol. 162 Jul. 1985 358.
`
`1393.
`
`2 of 139
`
`BI Exhibit 1136
`
`

`

`U.S. Patent
`
`Jun. 25, 1996
`
`Sheet 1 of55
`
`5,530,101
`
`I v L
`Q
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`FIGURE lB
`
`3 of 139
`
`BI Exhibit 1136
`
`

`

`U.S. Patent
`5,530,101
`D I V L T Q S P A S L A V S L G Q R A T
`_ V M T Q S P A T L S V S ? G � R A T
`_; S N L !:. S
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`G V P A R F S G S G F G T D F T L N
`P V E � E D T V T Y Y C Q H S W E I P Y
`T E' G G G T K L E I K
`FIGURE 2A
`· � V E S G G G L V K P G A S L � L
`E V Q � L E S G G G L V Q P G G S L R L
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`- v
`
`Jun. 25, 1996
`
`Sheet 2 of 55
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`E
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`FIGURE 2B
`
`4 of 139
`
`BI Exhibit 1136
`
`

`

`U.S. Patent
`
`Jun. 25, 1996
`
`Sheet 3 of 55
`
`5,530,101
`
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`41
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`FIGURE 3A
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`FIGURE 3B
`
`5 of 139
`
`BI Exhibit 1136
`
`

`

`U.S. Patent
`5,530,101
`D I V L T Q S P A S L A V S � G Q R A T
`
`Jun. 25, 1996
`
`Sheet 4 of 55
`
`D I Q M T Q S P S S L S A S V G D R V T
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`FIGURE 4A
`
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`FIGURE 4B
`
`M D V R S L T S E D S A V Y Y C A R G R
`
`P
`
`6 of 139
`
`BI Exhibit 1136
`
`

`

`U.S. Patent
`
`Jun. 2S, 1996
`
`Sheet 5 of SS
`
`5,530,101
`
`P G E K V T
`Q I V L T Q S P A I M S A S
`D I Q M m Q S P S S L S A S V G D R V T
`
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`FIGURE SA
`
`l
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`
`7 of 139
`
`BI Exhibit 1136
`
`

`

`U.S. Patent
`
`Jun. 25, 1996
`
`Sheet 6 of 55
`
`5,530,101
`
`D I V : T Q S P A T L S V 1 ? G D S V S
`
`E I V � T Q S P G T L S L S P G E R A r.-
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`8 of 139
`
`BI Exhibit 1136
`
`

`

`U.S. Patent
`
`Jun. 25, 1996
`
`Sheet 7 of 55
`
`5,530,101
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`9 of 139
`
`BI Exhibit 1136
`
`

`

`U.S. Patent
`
`Jun. 25, 1996
`
`Sheet 8 of 55
`
`5,530,101
`
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`10 of 139
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`BI Exhibit 1136
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`

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`11 of 139
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`12 of 139
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`BI Exhibit 1136
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`13 of 139
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`BI Exhibit 1136
`
`

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`14 of 139
`
`BI Exhibit 1136
`
`

`

`10
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`15 of 139
`
`BI Exhibit 1136
`
`

`

`U.S. Patent
`
`Jun. 25, 1996
`
`Sheet 14 of 55
`
`5,530,101
`
`I -
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`
`16 of 139
`
`BI Exhibit 1136
`
`

`

`U.S. Patent
`
`Jun. 25, 1996
`
`Sheet 15 of 55
`
`5,530,101
`
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`
`17 of 139
`
`BI Exhibit 1136
`
`

`

`U.S. Patent
`
`Jun. 25, 1996
`
`Sheet 16 of 55
`
`5,530,101
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`18 of 139
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`BI Exhibit 1136
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`
`y
`I
`y
`
`l
`
`V
`
`y
`I
`y
`
`K
`I
`K
`
`.IITIHnuUPIPSISI"anGIGGIGmSISS0wI.SISlevl“InnF
`IINIInsE0'0“MA5IIIvllvl
`
`FIGURE 16
`
`20 of 139
`
`BI Exhibit 1136
`
`20 of 139
`
`BI Exhibit 1136
`
`

`

`U.S. Patent
`
`Jun. 25, 1996-
`
`Sheet 19 of 55
`
`5,530,101
`
`TCTAGATGGGATGGAGCTGGATCTTTCTCTTCCTCCTGTCAGGTACCGCGGGCGTGCACT
`M G
` V H
`W
` S W I F
` L F L LS G T A
`G
`
`CTCAGGTCCAGCTTGTCCAGTCTGGGGCTGAAGTCAAGAAACCTGGCTCGAGCGTGAAGG
`L V 0 S G
`K
`S 0 V
`0
`AE V
`K
`P
` G S S V
`K
`
`TCTCCTGCAAGGCTTCTGGCTACACCTTTACTAGCTACAGGATGCACTGGGTAAGGCAGG
`R
`M
` H W V
` R 0
`VS C
`K
`
` A S G YT
`FT SY
`
`CCCCTGGACAGGGTCTGGAATGGATTGGATATATTAATCCGTCGACTGGGTATACTGAAT
`A
`P
`G
`
`0 G L E
`W
` I G YI N PST G YT
`E
`
`50
`110
`170
`230
`290
`350
`410
`
`20
`80
`140
`200
`260
`320
`380
`
`30
`90
`150
`210
`270
`330
`390
`
`40
`100
`160
`220
`280
`340
`400
`
`10
`70
`130
`190
`250
`310
`370
`430
`
`ACAATCAGAAGTTCAAGGACAAGGCAACAATTACTGCAGACGAATCCACCAATACAGCCT
`Y
`N
`Q
`K 0
`K A T I T A
`D
`N T A
`E S T
`K
`F
`
`ACATGGAACTGAGCAGCCTGAGATCTGAGGACACCGCAGTCTATTACTGTGCAAGAGGGG
`V Y Y C A
`R G
`Y M E
`R S E
`D T A
`L S S L
`
`GGGGGGTCTTTGACTACTGGGGCCAAGGAACCCTGGTCACAGTCTCCTCAGGTGAGTCCT
` 0 Y W G
`VS S
`G G
`V F
`0 G T
` L VT
`
`TAAAA CCTCTA GA
`
`FIGURE 17
`
`60
`120
`180
`240
`300
`360
`420
`
`21 of 139
`
`BI Exhibit 1136
`
`

`

`30
`TCTAGATGGAGACCGATACCCTCCTGCTATGGGTCCTCCTGCTATGGGTCCCAGGATCAA
`L L L W V l
`M
`E T D T
`L
`90
`
`70
`
`80
`
`100
`
`CCGGAGATATTCAGATGACCCAGTCTCCATCTACCCTCTCTGCTAGCGTCGGGGATAGGG
`T
`G D I QM
`T
`OSPSTLSASV
`G
`O
`R
`
`
`U.S. Patent
`
`Jon. 25, 1996
`
`Sheet 20 of 55
`
`5,530,101
`
`10
`
`20
`
`40
`
`60
`50
`L W V P G S
`110
`120
`
`130
`
`140
`
`150
`
`160
`
`170
`
`180
`
`TCACCATAACCTGCTCTGCCAGCTCAAGTATAAGTTACATGCACTGGTACCAGCAGAAGC
`I T
`CSASSS I SY
`M
`H W Y
`O
` OK
`V T
`
`190
`
`200
`
`210
`
`220
`
`230
`
`240
`
`CAGGCAAAGCTCCCAAGCTTCTAATTTATACCACATCCAACCTGGCTTCTGGAGTCCCTG
`PGK APKL L IYT
`TSNL ASG
`V P
`
`250
`
`am
`
`210
`
`280
`
`290
`
`300
`
`CTCGCTTCAGTGGCAGTGGATCTGGGACCGAGTTCACCCTCACAATCAGCTCTCTGCAGC
`0
`A
`R
` F SGS GS G TE FT LT
`IS SL
`
`310
`
`320
`
`330
`
`340
`
`350
`
`360
`
`CAGATGATTTCGCCACTTATTACTGCCATCAAAGGAGTACTTACCCACTCACGTTCGGTC
`F G
`Y Y C
`R S T
`F A T
`H
`Y P L T
`0
`
`P D 0
`370
`
`AGGGGACCAAGGTGGAGGTCAAACGTAAGTACACTTTTCTAGA
`QG TK VE VK
`
`380
`
`390
`
`400
`
`FIGURE 18
`
`22 of 139
`
`BI Exhibit 1136
`
`

`

`HES 12
`
`AG CTTC TAGATG GGATGG AG CT GGA TCTTT CTC TTCCTCC TG TCAG GT AC CG CGG G CG TG
`CACTCTCAGGTCCAGCTTGTCCAGTCTGGGGCTGAAGTCAAGAAACCTGGCTCGAGCGTG
`AAGGTC
`
`HES13
`
`CCCAGTCGACGGATTAATATATCCAATCCATTCCAGACCCTGTCCAGGGG CCTGCCTTAC
`CCAGTGCATCCTGTAGCTAGTAAAGGTGTAGCCAGAAGCCTTGCAGGAGACCTTCACGCT
`CGAGCCAGG
`
`HESl4
`
`TATA TT AA TC CG TCGA CTGG GTATA C TGAATACAATCAGAAG TTCAAGGA CAAGGCAA CA
`ATTACTGCAGACGAATCCACCAATACAGCCTACATGGAACTGAGCAGCCTGAGATCTGAG
`GACA
`
`HESIS
`
`ATATCGTCTAGAGGTTTTAAGGACTCACCTGAGGAGACTGTGACCAGGGTTCCTTGGCCC
`CAGTAGTCAAAGACCCCCCCCCCTCTTGCACAGTAATAGACTGCGGTGT CCTCAGATCTC
`AGGCTGCT
`
`FIGURE 19A
`
`HESl2
`
`HESl4
`
`HESl3
`
`FIGURE 19B
`
`HESl5
`
`•
`d
`•
`00
`�
`�
`g ......
`� ? N .. u. ..... \C \C QI\
`00 =­ti> ti> .. N ..... g, �
`Ul 'II Ul
`
`= 'II
`
`�
`�
`=
`�
`
`23 of 139
`
`BI Exhibit 1136
`
`

`

`JFDI CAAAT CTAGATGGAGACCGA TA CCCTCCTGCTATGGG TC C TCCTG CTATGGG TCCCAGGA
`TCAACCGGAGATATTCAGATGACCCAGTCTCCATCTACCCTCTCTGCTAGCGTCGGGGAT
`Jf02 ATAAATTAGAAGCTTGGGAGCTTTGCCTGGCTTCTGCTGGTACCAGTGCATGTAACTTAT
`ACTTGAGCTGGCAGAGCAGGTTATGGTGACCCTATCCCCGACGCTAGCAGAGAG
`JFD3 GCTCCCAAGCTTCTAATTTATACCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTC
`AGTGGCAGTGGATCTGGGACCGAGTTCACCCTCACAATCAGCTCTCTGCAGCCAGATGAT
`TTC
`JFD4 TATATCTAGAAAAGTGTACTTACGTTTGACCTCCACCTTGGTCCCCTGACCGAACGTGAG
`TGGGTAAGTACTCCTTTGATGGCAGTAATAAGTGGCGAAATCATCTGGCTGCAGAGAGCT
`GA
`FIGURE 20A
`
`JFDI
`
`JFD3
`
`JFD2
`HIND m
`FIGURE 20B
`
`I
`
`r:J'J.
`
`Cj
`•
`
`• � "'*' a
`�
`:= � I-" \C \C =-.
`rJ'J =­I'll � � � tll tll
`tll .... tll
`= .... �
`
`t.H
`
`=
`�
`
`JF04
`
`24 of 139
`
`BI Exhibit 1136
`
`

`

`U.S. Patent
`
`Jun. 25, 1996
`
`Sheet 23 of 55
`
`5,530,101
`lg Promoter
`Xbal�
`VHJ
`
`Eco RI
`
`Barn HI
`FIGURE 21
`
`lg Promoter
`XbaI�
`VJ
`
`pBR322
`
`PvuII
`Barn HI
`Hind ill
`Gp t
`FIGURE 22
`
`25 of 139
`
`BI Exhibit 1136
`
`

`

`U.S. Patent
`
`Jun. 2S, 1996
`
`Sheet 24 of SS
`
`60
`30
`ATGGATTTTCAAGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATACTGTCC
`M D F Q V Q
`_
`S F L L
`I
`S A
`S V
`
`5,530,101
`I L
`5
`
`!
`I V L T Q
`
`I Y D T
`
`L A
`
`120
`90
`AGAGGACAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCGTCTCCAGGGGCGA.�G
`? G E K
`R G Q
`S P A
`I M
`S A
`S
`180
`150
`GTCACCATGACCTGCAGTGGCAGCTCAAGTGTAAGTTTCATGTACTGGTACCAGCAGAGG
`V T M T C
`Y W Y Q Q R
`S F M
`S G S
`S
`S V
`240
`210
`CCAGGATCCTCCCCCAGACTCCTGATTTATGACACATCCAACCTGGCTTSTGGAGTCCCT
`S G V P
`S N
`P G
`S
`S P R L L
`300
`270
`GTTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCCGA.�TGGAG
`S G
`S G
`S G T
`S Y
`S L T
`I
`S R M E
`V R F
`360
`330
`GCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTACTTACCCGCTCACGTTCGGT
`Y P � T F G
`Y C 0 0 W
`S T
`
`A E D A A T Y
`A G T K L E � K FIGURE 23A
`M A V L G L L � C L V T F P
`S G ? G L V Q ?
`S V T
`S Y G V H W
`S T ) Y N A
`
`GCTGGGACCAAGCTGGAGCTGAAA
`
`60
`30
`ATGGCTGTCTTGGGGCTGCTCTTCTGCCTGGTGACATTCCCAAGCTGTGTCCTATCCCAG
`S C V L
`S Q
`120
`90
`GTGCAGCTGAAGCAGTCAGGACCTGGCCTAGTGCAGCCCTCACAGAGCCTGTCCATCACC
`V Q L K Q
`S Q
`S L
`S
`I T
`18C
`150
`TGCACAGTCTCTGGTTTCTCAGTAACAAGTTATGGTGTACACTGGATTCGCCAGTCTCCA
`C T V
`S G F
`I R Q
`S
`P
`240
`210
`GGAAAGGGTCTGGAGTGGCTGGGAGTGATATGGAGTGGTGGP..AGCACAGACTATAATGCA
`I W
`S G G
`
`G K G L E W � G V
`S � J �
`
`300
`270
`GCTTTCATATCCAGACTGACCATCAGCAAGGACAACTCCAAGAGCCAAGTTTTCTTTA.n.A.
`A
`_
`S � �
`"'
`-
`S
`K
`D
`N
`S
`!<
`S
`Q V F
`F !\
`360
`33G
`GTGAACAGTCTGC�.AC�TGCTGACACAGCCATATACTATTGTGCCAGAGCTGGGGACTAT
`A
`Y
`390
`A.ATTACGACGGTTTTGCTTACTGGGGCCAAGGGAC�CTGGTCACTGTCTCTGCG
`_
`
` I
`
`V �
`N Y ) G
`
`•
`
`•
`-
`v C A � � G D
`�
`Y W G Q G T L V
`- A
`FIGURE 23B
`
`v
`
`26 of 139
`
`BI Exhibit 1136
`
`

`

`U.S. Patent
`
`Jun. 25, 1996
`
`Sheet 25 of 55
`
`Eco RI
`
`Xbal
`
`5,530,101
`
`pVg1-dhfr
`
`Pvull
`
`FIGURE 24A
`
`EcoRI
`
`Xbal
`
`pVk
`
`FIGURE 24B
`
`27 of 139
`
`BI Exhibit 1136
`
`

`

`U.S. Patent
`
`Jun. 25, 1996
`
`Sheet 26 of 55
`
`5,530,101
`
`FIGURE 25
`
`28 of 139
`
`BI Exhibit 1136
`
`

`

`5,530,101
`S Q N V N A Y L N W Y Q Q K '='
`S '!' R E
`'
`S S � Q ?
`_
`
`I
`
`S S L Q P
`
`T
`
`21
`21
`
`41
`40
`
`61
`60
`
`81
`80
`
`101
`100
`
`l
`1
`
`21
`21
`
`� l
`� 1
`
`61
`60
`
`81
`80
`
`101
`99
`
`121
`�ls
`
`U.S. Patent
`
`Jun. 25, 1996
`
`Sheet 27 of 55
`
`D
`D
`
`I Q M T Q S P S S L S V S V G D R V T
`
`I Q M T Q S P S S L S A S V G D R V '!'
`
`A
`I T C Q
`I T C S G S S S V
`
`S F M Y W Y Q Q K ?
`
`G L
`G K
`
`A
`A
`
`A
`I Y G
`P K L L
`P K L L I Y D '"" S N L
`
`A
`
`A G V P :;
`S G V P S
`
`R F S G S G S G T D c T F '!'
`
`R F S G S G S G '!' D v T
`
`T Y Y C Q Q Y N N W ? ? '!' F G C
`T Y Y C Q Q W S T Y P L "' F G Q
`G T K V E V K
`G T K V E V K
`
`E D
`E D
`
`I
`I
`
`A
`A
`
`FIGURE 26A
`
`A V Q L L E S G G G L V Q P G G S L R -
`E V Q L L E S G G G L V Q P G G S L R �
`
`S C
`S C
`
`A
`A
`
`A
`A
`
`S G F T
`S G F T
`
`F S
`
`A
`
`S
`
`A M S
`
`w v
`
`? G K G
`
`"1 V £._
`
`A
`N
`
`D S V N G R F T
`A
`A
`F
`
`. .
`
`,_,
`._,
`
`I S R N D S K N "' L Y
`
`V 'T' S Y G V �
`= __;_ -=-----=------'�-� w v
`'
`- W S � � � "" !)
`P G :< G L � W V
`A W :< Y � N G N ) K
`_
`"' � Y
`·v
`I Y Y C A R D �
`I S R F T
`I S R D N S K N
`G ? Y V S 0 T F
`_ A E W G Q G '" � V "'
`;; R __ A_
`_ A Y W G Q G '!' - V T V
`I '1. Y C
`v·
`
`L Q M N G L Q
`L Q M N S L Q
`
`A
`A
`
`Z V S
`::. D T
`
`A
`A
`
`G D Y
`
`N Y D G
`
`s s
`s
`s
`
`FIGURE 26B
`
`29 of 139
`
`BI Exhibit 1136
`
`

`

`Jun. 25, 1996
`
`Sheet 28 of 55
`
`5,530,101
`
`U.S. Patent
`
`vcl3
`
`60
`50
`4 0
`30
`20
`1 0
`TTCTGCTGGT ACCAGTACAT GAAACTTACA CTTGAGCTGC CACTGCAGGT GATGGTGACG
`
`100
`80
`70
`90
`CGGTCACCCA CTGAGGCACT GAGGCTAGAT GGAGACTGGG TCATTTG
`
`vcl4
`
`60
`5 0
`4 0
`3 0
`2 0
`10
`CATGTACTGG TACCAGCAGA AGC.CAGGAAA AGC.TCCGAAA CTTCTGATTT ATGACACATC
`
`120
`llO
`100
`90
`80
`7 0
`CAACCTGGCT TCTGGAGTCC CTTCCCGCTT CAGTGGCAGT GGGTCTGGGA CCGATTACAC
`
`130
`CTTTACAATC TCTTCA
`
`vclS
`
`60
`4 0
`30
`20
`1 0
`so
`TGTGTCTAGA AAAGTGTACT TACGTTTTAC CTCGACCTTG GTCCCTTGAC CG.�GTGAG
`
`120
`1 10
`100
`80
`70
`90
`CC-GGTAAGTA CTCCACTGCT GGCAGTAATA AGTGGCTATA TCTTCCGGCT GAAGTGAAGA
`
`130
`GATTGTAAAG GTGT�-�T
`
`vcl6
`
`60
`50
`4 0
`20
`10
`30
`CACATCTAGA CCACCATGG;.. TTT�C.r..AGTG CAGATCTTCA GCT!CCTGCT AATC.�GTGCC
`
`70
`80
`90
`100
`TCAGTCATAC TGTCCAG.AGG �GATATTCAA ATGACCCAGT CTCCATCT
`
`FIGURE 27A
`
`30 of 139
`
`BI Exhibit 1136
`
`

`

`Jun. 25, 1996
`
`Sheet 29 of 55
`
`5,530,101
`
`U.S. Patent
`
`vcll
`
`so
`60
`40
`30
`20
`10
`TAGTCTGTCG ACCCACCACT CC.�TATCACT CCCACCCACT CGAGTCCCTT TCCAGGAGCC
`1 10
`120
`80
`70
`100
`90
`TGGCGGACCC AGTGTACACC ATAACTTGTT ACGGTGAAAC CACTGGCGGC ACAAGACAGT
`
`130
`CTCAGAGATC CTCCTGGC
`
`vcl2
`
`10
`60
`50
`40
`20
`30
`TGGTGGGTCG ACAGACTATA ATGCAGCTTT CATATCCAGA TTTACCATCA GCAGAGACAA
`
`70
`110
`80
`120
`100
`90
`CAGCAAGAAC ACACTGTATC TCCAAATGAA TAGCCTGCA A GCCGAGGACA CAGCCATATA
`
`TTATTG
`
`wps54
`
`so
`10
`60
`40
`30
`20
`AC..�CTCTAGA CCACCATGGC TGTCTTGGGG CTGCTCTTCT GCCTGGTGAC ATTCCCAAGC
`
`100
`80
`70
`120
`110
`90
`TGTGTCC7AT CCC-CTGTCCA GCTGCTAGAG AGTGGTGGCG GTCTGGTGC.� GCCAGGAGGA
`
`130
`TCTCTGAGAC
`
`wps57
`
`20
`10
`60
`50
`40
`30
`ACACTCTAGA AGTTAGGACT C�CCTGAAGA GACAGTGACC AGAGTCCCTT GGCCCCAGTA
`
`110
`80
`70
`100
`90
`AGCJV:iAA.CCG TCGTA.�TTAT �GTCCC�..GC TCTGGCACAA T.l\ATATATGG C�GTGTCC
`
`FIGURE 27B
`
`31 of 139
`
`BI Exhibit 1136
`
`

`

`U.S. Patent
`
`Jun. 2S, 1996
`
`Sheet 30 of SS
`
`5,530,101
`
`0.07 -.,...--------------------�
`
`0.06
`
`0.05
`
`UJ UJ 0.04
`a: u. ..... Q z �
`0 ID 0.03
`
`0.02
`
`0.01
`
`.01
`
`0.00 --,_., ----------------------J
`FIGURE 28
`
`.1
`100
`10
`1
`COMPET ITOR AN TIBODY CONCENTRATION (nM)
`
`1000
`
`32 of 139
`
`BI Exhibit 1136
`
`

`

`U.S. Patent
`
`Jun. 25, 1996
`
`Sheet 31 of 55
`
`5,530,101
`
`UJ � a:
`0 2000
`0 CL a:
`0 (.) z
`:E CL (.)
`
`1000
`
`. 1
`
`10