United States Patent
`Carter et al.
`
`r19J
`
`111111111111111111111111111111111111111111111111111111111111111111111111111
`US00582133 7 A
`[11] Patent Number:
`[45] Date of Patent:
`
`5,821,337
`Oct. 13, 1998
`
`(54] IMMUNOGLOBULIN VARIANTS
`lnventors: Paul J. Carter; Leonard G. Presta,
`both of San Francisco, Calif.
`
`[75]
`
`[73] As.5ignee: Genentecb, Inc., South San Francisco,
`Calif.
`
`[21] Appl. No.: 934,373
`
`[22] Filed:
`
`Aug. 21, 1992
`
`Related U.S. Application Data
`
`continuation-in-part of Ser. No. 715,272, Jun. 14, 1991,
`
`(63] Continua tion-in-par t of PCT/US92/05126 Jun. 15, 1992
`
`abandoned.
`lnt. Cl.6 ...••..••..•...•..................•...•..••..••...... C07K 16/00
`[51]
`[52] U.S. Cl .................... 530/387.3; 530/350; 530/388.2;
`424/133.1
`[58] Field of Search
`................................. 530/387.3, 350,
`530/388.2; 424/133.1
`
`[56]
`
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`
`Primary Examiner-Lila Feisee
`Assis/a!// Examiner-Minh-Tam Davis
`Attorney, Agent, or Firm-Wendy M. ll.ee
`[57]
`ABSTRACT
`Variant immunoglobuJins, particularly humanized a ntibody
`polypeptides are provided, along with methods for their
`preparation and use. Consensus immuooglobulin sequences
`and s1ruc1ural models are also provided .
`15 Claims, 12 Drawing Sheets
`
`
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`1 of 76
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`bodies 10 IgE" International Archives of Allergy and 111111111-
`nology J 07(1-3):308-312 (May 1995).
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`nology 15 1(4):2296-2308 (Aug. 1993).
`Smith-Gill et al., "A Three-dimensional Model of an
`Anli-lysozyme Antibody" Mo!. Biol. 194:713-724 (1987).
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`and treatmcnl of disease" Clin. Exp. l1111111mol. 79:315-321
`(1990).
`Stanford, "A Predictive Method for Determining Possible
`Three-dimensional Foldings of lmmunoglobulin Backbones
`Theor. Biol.
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`Antibody, fm1111mo Radiop!tarm 2:1-13 (1989).
`Tempest et al., "Reshaping a Human Monoclonal Antibody
`to Inhibit Human Respiratory Syncylial Virus infection Io
`Vivo'' Bio/Technology 9: 266-271 (Mar. 1991).
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`Treated with Anti-IL-2 Receptor Monoclonal antibody
`,
`. 1i·ansplantation 45(1):226-228 (Jan. 1988).
`Campa1b-6
`Verhoeyen & Riecbmann, "Engineering of Antibodies"
`BioEssays 8(2):74-78 (Feb./Mar. 1988).
`
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`bodies" Protein Struc//lre, Folding, and Design 2 (Proc.
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`1987), Dale L. Oxender, New York:Alan R. Liss, Inc. pp.,
`501-502 (1987).
`
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`Epenetos, lsi edition, Chapman & llall Medical pp. 37-43
`(l991).
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`Domains i Esclterchia Coli: Isolation of Antigen Binding
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`VJJ:1144-115 1 (1989).
`
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`immunoglobulin variable domains secreted from Escheri­
`chia coli .. Nmure 34 J :544-546 (1989).
`
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`tion-Associated Ao1igen (LFA)-1 Monoclonal Antibody
`and Rcengioceriog of tbe Humanized An1ibody for Binding
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`
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`0 (Syrop. on Protein and Pharm. Eng. Mol. Cell. Biol. Park
`Ci1y, Utah) 13 Pan A:96 (1989).
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`ies" Investigation and Exploitation of Artlibody Combining
`Sites, Eric Reid, Plenum Press pp. 139-140 (1985 ).
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`Blocker e1 al., VCH pp. 189-197 (1987).
`,
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`Lond. B 324:99-109 (1 989).
`
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`Transfer of Immune Modulation Properties and ldiotype
`Exprcs.sion" J. of Immunology 148(9):2756-2763 (May
`1992).
`
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`Involves Framework Residues"
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`(manuscripl) 1996, 37 pps.
`
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`anlibody for 1be therapy of solid tumors and other disorders"
`Cancer Researc!t (in prcs.s) pp. 1-32 (1997) oow published
`57(20):4593-4599 (Oc1. 1.5, 1997).
`
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`No. 451,261 Bl (Oct. 22, 1996) 10 pages.
`
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`lute, Abstrac1) (May 1987) Br. J. Cancer 56:509.
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`Vcrhoeycn el al., "Humanising Mouse An1ibodics: A Pro1ein
`Engineeriog Approach" Soc. for Analytical Cytology (Xlltb
`ln11. Mtg. for tbe Soc. for Analytical Cytology, Cambridge,
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`
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`
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`
`
`
`4 of 76
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`

`FIG. 1A
`
`405
`
`HU405
`
`HUVLKI
`
`50
`40
`30
`20
`10
`OIVMTQSHKFMSTSVGORVSITCKASQDVNTAVAWYQQKPGHSPKLLIYSASFRYT
`I
`I I I I I
`I
`I I
`I I I
`I I I I
`I I I
`I
`I
`I
`I I
`OIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLES
`II II
`I I
`I I
`II II
`DIQMTQSPSSLSASVGDRVTITCRASQDVSSYLAWYQQKPGKAPKLLIYAASSLES
`
`I
`I
`
`VL-CDRl
`
`VL-CDR2
`
`405
`
`HU405
`
`HUVLl(I
`
`100
`90
`80
`70
`60
`GVPDRFTGNRSGTDFTFTISSVQAEDLAVYYCQQHYTTPPTFGGGTKLEIKRA
`I
`I
`I I
`I
`I
`I
`I
`I
`I
`I
`I
`I I
`I
`I I
`I I
`I
`I
`I
`GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRT
`I I I I
`I
`I
`I
`I II I I
`GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSLPYTFGQGTKVEIKRT
`
`VL-CDR3
`
`•
`
`� • 'J:J
`� � ..... �
`
`=
`.....
`
`0
`fl
`.....
`��
`
`..... IC IC QO
`
`.....
`
`"1 :r ("!) � .....
`0 � ..... N
`
`VI ... 00 N
`� ... �
`�
`-.....l
`
`
`
`5 of 76
`
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`

`FIG 18
`
`405
`
`HU405
`
`HUV HIII
`
`40
`30
`20
`10
`50 A
`EVQLQQSGPELVKPGASLKLSCTASGFNIKOTYIHWVKQRPEQGLEWIGRIYPTN
`I
`I
`I I
`I I
`I I
`I I
`I
`I
`I
`I
`I
`I
`I I
`I I
`I I
`I
`I
`I
`I
`I I
`EVQLVESGGGLVQPGGSLRLSCAA SGFNIKDTYIHWVRQAPGKGLEWVARIYPTN
`I I I
`I I I I
`I
`I I I I
`I I I
`I I I I
`I
`I I I I
`EVQLVESGGGLVQPGGSLRLSCAASGFTFSOYAMSWVRQAPGKGLEWVAVISENG
`
`Vff-CDR1
`
`Vu-CDR2
`
`405
`
`HU405
`
`HUV HIII
`
`90
`80 ABC
`70
`60
`lOOABC
`GYTRYOPKFQOKATITAOTSSNTAYLQVSRLTSEOTAVYYCSRWGGOGFYAMDYW
`I I I I I I I I
`I I I
`I I
`I
`I
`I
`I I I
`I I
`I I I I I I I I
`I
`I
`I
`GYTRYAOSVKGRFTISADTSKNTAYLQMNSLRAEOTAVYYCSRWGGDGFYAMDVW
`I I I I I I
`I
`I I
`I
`I
`I
`I I
`I
`I
`I
`I
`I
`I I
`I l I II I
`I I
`I
`SOTYYAOSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCARORGGAVSYFOVW
`
`405
`
`HU4D5
`
`110
`GQGASVTVSS
`I I
`I I
`GQGTLVTVSS
`
`HUVHIII
`
`GQGTLVTVSS
`
`Vu-CDR3
`
`.
`
`d • rJ).
`� � � �
`
`=
`�
`
`0
`(') !""'
`.....
`��
`.... \C \C 00
`
`Cl) er � � .... N
`0 �
`..... N
`
`01
`.... oe N
`�
`.... w
`w
`....._J
`
`
`
`6 of 76
`
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`IPR2017-01374
`
`

`

`U.S. Patent
`
`Oct. 13, 1998
`
`Sheet 3of 12
`
`5,821,337
`
`Anneal huVL or huVH oligomers
`to pAKl template
`3·---A----A----A----A----A-� 5'
`_________ ....,. __________ ____ _
`/
`1. Ligate
`2. Isolate assembled oligomers
`(Xhol-; Stu/+)
`3. Anneal to pAKl template
`4. Extend and ligate
`
`l. Transform E. coli
`2. Isolate
`phagemid pool
`3. Enrich for huVL and huVH(Xho I "t; Stu/-)
`4. Sequence verify
`
`Xho/
`
`FIG. 2
`
`pAK2
`
`
`
`7 of 76
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`

`U.S. Patent
`
`Oct. 13, 1998
`
`Sheet 4 of12
`
`5,821,337
`
`100
`
`- c::
`8 0 80
`... ·-c -0 �
`() .... ....... � 0;.::::
`- 8
`5 Q.
`(.) - 60
`�o 0.. ()
`
`40
`
`huMAb4D5-8
`
`muMAb4D5
`
`4
`
`8
`12
`[MAb4D5 variant] µg/ml
`
`16
`
`FIG. 3
`
`
`
`8 of 76
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`

`U.S. Patent
`
`Oct. 13, 1998
`
`Sheet 5of 12
`
`5,821,337
`
`FIG 4
`
`
`
`9 of 76
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`

`U.S. Patent
`
`Oct. 13, 1998
`
`Sheet 6of 12
`
`5,821,337
`
`VL
`40
`30
`10
`20
`muxCD3
`OIQMTQTTSSLSASLGDRVTISCRASQOIRNYLNwYQQKP
`**
`•
`*
`huxC03vl
`DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKP
`# # #
`hUKI
`
`DIQMTQSPSSLSASVGORVTITCBASOSISNYI.AWY
`QQKP6
`AAAAAAAA
`CDR-Ll
`
`so
`70
`80
`60
`muxCD3
`
`DGTVKLLIYYTSRLHSGVPSKFSGSGSGTDYSLTISNLEQ
`*
`*
`* **
`*
`****
`huxCD3vl
`GKAPKLLIYYTSRLESGVPSRFSGSGSGTOYTLTISSLQP
`## #
`#
`huKI
`GKAPKLLIYAASSLESGVPSRFSGSGSGTDFTLTISSLQP
`,..,..,..
`CDR-L2
`
`90
`100
`muxC03
`EDIATYFCQQGNTLPWTFAGGTKLEIK
`*
`*
`**
`*
`huxCD3vl
`
`EOFATYYCQQGNTLPWTFGQGTKVEIK
`# #
`hul(I
`EDFATYYCOOYNSLPWT
`FGQGTKVEIK
`
`Vu muxC03
`40
`20
`10
`30
`EVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNwvKQS
`** ** * * *** *
`* *
`huxcoJvl
`EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQA
`I# ## # #
`huIII
`EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAHSWVRQA
`
`60
`50
`70
`muxCD3
`HGKNLEWMGLINPYKGVSTYNQKFKDKATLTVDKSSSTAY
`* *
`**
`* **** ** **
`**
`huxCDJvl
`
`PGKGLEWVALINPYKGVTTYAOSVKGRFTISVDKSKNTAY
`## #### # #
`# #
`I
`HuIII
`PGKGLEWVSVISGOGGSTYYADSYKG
`RFTISRDNSKNTLY
`AAAA
`CDR-H2
`
`lOOabcde 110
`90
`80 abc
`muxCD3
`TVTVSS
`MELLSLTSEDSAVYYCARSGYYGDSOWYFOVWGAGT
`* *
`**** ** *
`huxCOJvl
`LQMNSLRAEDTAVYYCARSGYYGOSOWYFDVWGQGTLVTVSS
`########### #
`huIII
`TVSS
`LQMNSLRAEDTAVYYCARGRVGYSLSGLYDYWGQGTLV
`p ET S
`AAAAAAAAAAA
`COR-HJ
`
`FIG. 5
`
`
`
`10 of 76
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`

`H52H4-160
`
`pH52-8.0
`
`FIG. 6A-1
`
`30
`10
`20
`QVQLQQSGPELVKPGASVKISCKTSGYTFTE
`·*** ·** **·**·*···** ********
`MGWSCIILFLVATATGVHSEVQLVESGGGLVQPGGSLRLSCATSGYTFTE
`10
`20
`30
`40
`50
`
`H52H4-160
`
`pH52-8.0
`
`80
`70
`60
`50
`40
`YTMHWMKQSHGKSLEWIGGFNPI<NGGSSHNQRFMDKATLAVDKSTSTAYM
`******·*· **·***··*·******·********· *··**********
`YTMHWMRQAPGKGLEWVAGINPKNGGTSHNQRFMDRFTISVDKSTSTAYM
`60
`100
`70
`80
`90
`
`H52H4-160
`
`_ pH52-8. 0
`
`130
`120
`110
`100
`90
`ELRSLTSEDSGIYYCARWRGLNYGFDVRYFDVWGAGTTVTVSSASTKGPS
`. . ** ·**···********************** ** ************
`QMNSLRAEDTAVYYCARWRGLNYGFDVRYFDVWGQGTLVTVSSASTKGPS
`110
`120
`130
`140
`150
`
`H52H4-160
`
`pH52-8.0
`
`180
`170
`160
`150
`140
`VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
`****** *·*** ·************************************
`VFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
`160
`170
`180
`190
`200
`
`H52H4-160
`
`pH52-8.0
`
`H52H4-160
`
`pH52-8.0
`
`230
`220
`210
`200
`190
`QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH
`************** **··***** ***·********** ** * *
`QSSGLYSLSSVVTVTSSNFGTQTYTCNVDHKPSNTKVDKTVERKCC---V
`210
`230
`220
`240
`240
`250
`270
`280
`260
`TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK
`******* ··*************************************·
`ECPPCPAPP-VAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQ
`250
`260
`270
`280
`290
`
`•
`
`0
`• 00
`� � ..... n>
`
`=
`.....
`
`0
`(") !""'
`....
`��
`.... \0 �
`
`00 ==­� � .....
`0 ..., .... N
`
`--.l
`
`(JI ,,.. oe N
`� ,,.. w
`w
`.....:.
`
`
`
`11 of 76
`
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`IPR2017-01374
`
`

`

`FIG 6A-2
`
`H52H4-160
`
`pH52-8.0
`
`330
`320
`310
`300
`290
`FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS
`*******· *************·***·********·***************
`FNWYVDGMEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVS
`300
`310
`320
`330
`340
`
`HS2H4-160
`
`pH52-8.0
`
`H52H4-160
`
`pH52-8.0
`
`HS2H4-160
`
`pH52-8.0
`
`380
`370
`340
`360
`350
`NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP
`**·***********·***********************************
`NKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP
`350
`360
`370
`380
`390
`
`430
`420
`410
`400
`390
`SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
`**********************·***************************
`SDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFS
`400
`410
`420
`430
`440
`
`450
`440
`CSVMHEALHNHYTQKSLSLSPGK
`***********************
`CSVMHEALHNHYTQKSLSLSPGK
`460
`450
`
`•
`
`0
`• rJ'l
`� � ..... �
`
`=
`.....
`
`0
`�
`......
`�(;)
`
`...... '° '° 00
`
`00 =­� r> ...
`0 ....., ...... N
`
`00
`
`Ul ... QC
`N
`� ... (M
`(M
`....:a
`
`
`
`12 of 76
`
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`

`

`RG. 68
`
`H52L6-158
`
`10
`30
`20
`DVQMTQTTSSLSASLGDRVTINCRASQDINN
`*·****· ******·****** *********
`pH52-9.0 MGWSCIILFLVATATGVHSDIQMTQSPSSLSASVGDRVTITCRASQDINN
`10
`20
`30
`40
`50
`
`80
`70
`60
`50
`40
`HS2L6-158 YLNWYQQKPNGTVKLLIYYTSTLHSGVPSRFSGSGSGTDYSLTISNLDQE
`*********
`• ***************************·****·*· *
`pH52-9.0 YLNWYQQKPGKAPKLLIYYTSTLHSGVPSRFSGSGSGTDYTLTISSLQPE
`60
`70
`80
`90
`100
`
`130
`120
`110
`100
`90
`H52L6-158 DIATYFCQQGNTLPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS
`*·***·************ *•·····························
`pH52-9.0 DFATYYCQQGNTLPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS
`110
`120
`130
`140
`150
`
`150
`140
`180
`170
`160
`H52L6-158 VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL
`••••••••••••••••••••••••••••••••••••••••••••••••••
`pHS2-9.0 VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL
`160
`170
`180
`190
`200
`
`H52L6-158
`
`pH52-9.0
`
`210
`200
`190
`SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
`•••••••••••••••••••••••••••••••••
`SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
`230
`220
`210
`
`d
`• IJ1
`.
`� � � �
`
`=
`�
`
`0
`(') �
`.....
`�w
`..... � � 00
`
`00 =­� � ...
`0 � ..... N
`
`�
`
`tll -.. QC
`N
`.......
`-.. w
`w
`'-l
`
`
`
`13 of 76
`
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`
`

`

`U.S. Patent
`
`Oct. 13, 1998
`
`Sheet 10 of 12
`
`5,821,337
`
`FIG. 7A-1
`
`verA.hcfabl
`verI.hcfab2
`verN. hcfab!
`
`lEVQLVBSGGGLVQPGGSLRLSCATSGYTFTEYTMHWMRQAPGKGLBWVAG
`1 EVQLVESGGGLVQPGGSLRLSCATSGYTFTEYTMHWMRQAPGKGLBWVAG
`1 EVQLVESGGGLVQPGGSLRLSCATSGYTFTEY'IMHWMRQAPGKGLEWVAG
`verO.hcfab
`1 EVQLVBSGGGLVQPGGSLRLSCATSGYTFTEYTMHWMRQAPGKGLBWVAG
`verO.hcfab25
`1 EVQLVBSGGGLVQPGGSLRLSCATSGYTFTEY'IMHWMRQAPGKGLBWVAG
`verP. hcfab 6
`1 EVQLVBSGGGLVQPGGSLRLSCATSGYTFTBYTMHWMR.QAPGKGLEWVAG
`verR.hcfab�
`verQ. hcfab 7
`1 EVQLVBSGGGLVQPGG SLRLSCATSGYTFTBY'IMHWMRQAPGKGLEWVAG
`1 EVQLVBSGGGLVQPGG SLRLSCATSGYTFTBY'IMHWMRQAPGKGLBWVAG
`vers. hcfab10
`1 EVQLQQSGPBLVQPGGSLRLSCATSGYTFTBYTMHWMRQAPG
`KGLEWVAG
`verT .hcfab
`1 EVQLV