- 33 -
`
`region of the variable region from the Aval site
`and to include the 5' residues of the human
`constant region up to and including a unique Narl
`site which had been previously engineered into the
`constant region.
`A Hindlll site was introduced to act as a marker
`for insertion of the linker.
`The linker was ligated to the VL fragment and the
`413 bp EcoRl-Narl adapted fragment was purified
`from the ligation mixture.
`The constant region was isolated as an Narl-BamHl
`fragment from an M13 clone NW361 and was ligated
`with the variable region DNA into an
`EcoRl/BamHl/ClP pSP65 treated vector in a three
`way reaction to yield plasmid JA143.
`Clones were
`isolated after transformation into E.coli and the
`linker and junction sequences were confirmed by
`the presence of the Hindlll site and by DNA
`sequencing.
`LIGHT CHAIN GENE CONSTRUCTION - VERSION 2
`The construction of the first chimeric light chain
`gene produces a fusion of mouse and human amino
`acid sequences at the variable-constant region
`junction.
`In the case of the OKT3 light chain
`the amino acids at the chimera junction are:
`•.•••..• Leu-Glu-Ile-Asn-Arg/
`-/Thr-Val-Ala
`VARIABLE
`CONSTANT
`-- -- -- ---- ---~---- · This arrangement of sequence introduces a
`
`9.2
`
`potential site for Asparagine (Asn) linked
`(N-linked) glycosylation at the V-C junction.
`Therefore, a second version of the chimeric light
`chain oligonucleotide adapter was designed in
`which the threonine (Thr), the first amino acid of
`the human constant region, was replaced with the
`equivalent amino acid from the mouse constant
`region, Alanine (Ala).
`
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`

`- 34 -
`
`An internal Hindlll site was not included in this
`adapter, to differentiate the two chimeric light
`chain genes.
`The variable region fragment was isolated as a 376
`bp EcoRl-Aval fragment.
`The oligonucleotide
`linker was ligateq to Narl cut pNW361 and then the
`adapted 396bp constant region was isolated after
`recutting the modified pNW361 with EcoRl.
`The
`variable region fragment and the modified constant
`region fragment were ligated directly into
`EcoRl/ClP treated pEE6hCMVneo to yield pJA137.
`Initially all clones examined had the insert in
`Therefore, the insert
`the incorrect orientation.
`was re-isolated and recloned to turn the insert
`round and yield plasmid pJA141.
`Several clones
`with the insert in the correct orientation were
`obtained and the adapter sequence of one was
`confirmed by DNA sequencing
`HEAVY CHAIN GENE CONSTRUCTION
`CHOICE OF HEAVY CHAIN GENE ISOTYPE
`The constant region isotype chosen for the heavy
`chain was human IgG4.
`GENE CONSTRUCTION
`The heavy chain cDNA sequence showed a Banl site(sei:lD""°:~
`near the 3' end of the variable region (Fig. 2(a)].
`/1
`The majority of the sequence of the variable
`region was isolated as a 426bp. EcoRl/ClP/Banl
`An oligonucleotide adapter was
`fragment.
`designated to replace the remainder of the 3'
`region of the variable region from the Banl site
`up to and including a unique BindIII site which
`had been previously engineered into the first two
`amino acids of the constant region.
`The linker was ligated to the VH fragment and the
`EcoRl-Hindlll adapted fragment was purified from
`the ligation mixture.
`
`~
`
`9.3.
`9.3.1.
`
`9.3.2.
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`

`- 35 -
`
`The variable region was ligated to the constant
`region by cutting pJA91 with EcoRl and Hindlll
`removing the intron fragment and replacing it with
`the VH to yield pJA142.
`Clones were isolated
`after transformation into E.coli JMlOl and the
`linker and junction sequences were confirmed by
`DNA sequencing.
`(N.B. The Hindlll site is lost
`on cloning) .
`
`CONSTRUCTION OF CHIMERIC EXPRESSION VECTORS
`neo AND gpt VECTORS
`The chimeric light chain (version l) was removed
`from pJA143 as an EcoRl fragment and cloned into
`EcoRl/ClP treated pEE6hCMVneo expression vector to
`yield pJA145.
`Clones with the insert in the
`correct orientation were identified by restriction
`mapping.
`The chimeric light chain (version 2) was
`constructed as described above.
`The chimeric heavy chain gene was isolated from
`pJA142 as a 2.SKbp EcoRl/BamHl fragment and cloned
`into the EcoRl/Bcll/ClP treated vector fragment of
`a derivative of pEE6hCMVgpt to yield plasmid
`pJA14 4.
`GS SEPARATE VECTORS
`GS versions of pJA141 and pJA144 were constructed
`by replacing the neo and gpt cassettes by a
`BamBl/Sall/ClP treatment of the plasmids,
`isolation of the vector fragment and ligation to a
`GS-containing fragment from the plasmid pR049 to
`yield the light chain vector pJA179 and the heavy
`chain vector pJA180.
`GS SINGLE VECTOR CONSTRUCTION
`Single vector constructions containing the cL
`(chimeric light), cH (chimeric heavy) and GS genes
`on one plasmid in the order cL-cH-GS, or cH-cL-GS
`
`10.
`10 .1.
`
`10.2.
`
`10.3.
`
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`

`11.
`11.1.
`
`- 36 -
`
`and with transcription of the genes being head to
`tail e.g. cL>cH>GS were constructed.
`These
`plasmids were made by treating pJA179 or pJA180
`with BamHl/ClP and ligating in a Bglll/Hindlll
`hCMV promoter cassette along with either the
`Hindlll/Bamlil fragment from pJA141 into pJA180 to
`give the cH-cL-GS plasmid pJA182 or the
`Hindlll/BamHl fragment from pJA144 into pJA179 to
`give the cL-cH-GS plasmid pJA181.
`
`EXPRESSION OF CHIMERIC GENES
`EXPRESSION IN COS CELLS
`The chimeric antibody plasmid pJA145 (cL) and
`pJA144 (cH) were co-transfected into COS cells and
`supernatant from the transient expression
`experiment was shown to contain assembled antibody
`which bound to the HUT 78 human T-cell line.
`Metabolic labelling experiments using 35s
`methionine showed expression and assembly of heavy
`and light chains.
`However the light chain ·
`mobility seen on reduced gels suggested that the
`potential glycosylation site was being
`glycosylated.
`Expression in COS cells in the
`presence of tunicamycin showed a reduction in size
`of the light chain to that shown for control
`chimeric antibodies and the OKT3 mouse light
`chain.
`Therefore JA141 was constructed and
`expressed.
`In this case the light chain did not
`show an aberrant mobility or a size shift in the
`presence or absence of tunica.mycin.
`This second
`
`version of the chimeric light chain, when
`expressed in association with chimeric heavy (cH)
`chain, produced antibody which showed good binding
`to HUT 78 cells.
`In both cases antigen binding
`was equivalent to that of the mouse antibody.
`
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`

`11.2
`
`12.
`
`12. 1.
`
`- 37 -
`
`EXPRESSION IN CHINESE HAMSTER OVARY (CHO} CELLS
`Stable cell lines have been prepared from plasmids
`PJA141/pJA144 and from pJA179/pJA180, pJA181 and
`pJA182 by transfection into CHO cells.
`
`CDR-GRAFTING
`The approach taken was to try to introduce
`sufficient mouse residues into a human variable
`region framework to generate antigen binding
`activity comparable to the mouse and chimeric
`antibodies.
`VARIABLE REGION ANALYSIS
`From an examination of a small database of
`structures of antibodies and antigen-antibody
`complexes it is clear that only a small number of
`antibody residues make direct contact with
`antigen. Other residues may contribute to
`antigen binding by positioning the contact
`residues in favourable configurations and also by
`inducing a stable packing of the individual
`variable domains and stable interaction of the
`light and heavy chain variable domains.
`The residues chosen for transfer can be identified
`in a number of ways:
`By examination of antibody X-ray crystal
`(a)
`structures the antigen binding surf ace can
`be predominantly located on a series of
`loops, three per domain, which extend from
`the B-barrel framework.
`By analysis of antibody variable domain
`sequences regions of hypervariability
`[termed the Complementarity Determining
`Regions (CDRs) by wu and Kabat (ref.- 5)]
`can be identified.
`In the most but not
`all cases these CDRs correspond to, but
`extend a short way beyond, the loop regions
`noted above ..
`
`(b)
`
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`

`- 38 -
`
`{c)
`
`Residues not identified by {a) and (b) may
`contribute to antigen binding directly or
`indirectly by affecting antigen binding
`site topology, or by inducing a stable
`packing of the individual variable domains
`and stabilising the inter-variable domain
`interaction.
`These residues may be
`identified either by superimposing the
`sequences for a given antibody on a known
`structure and looking at key residues for
`their contribution, or by sequence
`alignment analysis and noting
`"idiosyncratic" residues followed by
`examination of their structural location
`and likely effects.
`12 • l . 1. LIGHT CHAlJb T~ NO?'t1i And. "l I
`'
`.
`l
`.
`Figure 3Ashows an alignm1~!Div~e.%'f~nces for the
`human framework region ~l and the OKT3 light
`(.$UJ.~~l)·.~
`"
`variable region.
`The
`ructural loops (LOOP) and
`.I\
`CDRs (KABAT) believed to correspond to the antigen
`binding region are marked. Also marked are a
`number of other residues which may also contribute
`to antigen binding as described in 13.l~·i 1 )
`(5~ -XD NQ:
`1ftllC\~
`Above the sequence in Figure 3A_the resi
`type
`indicates the spatial location of each residue
`side chain, derived by examination of resolved
`structures from X-ray crystallography analysis.
`The key to this residue type designation is as
`follows:
`N - near to CDR (From X-ray Structures)
`P - Packing
`B - Buried Non-Packing
`s
`Surf ace
`Exposed
`E
`* - Interface
`I - Interface
`- Packing/Part Exposed
`? - Non-CDR Residues which may require to be left
`as Mouse sequence.
`
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`

`(
`
`- 39 -
`
`'1/
`,.~
`LS'C'Q -ro ('l.O: ~~ f(d
`Residues underlined in Figure 3,., are amino acids.
`LS~Q J:~ Y'ID: i A'rd 'Tl
`/'
`RElAwas chosen as the human framework because the
`light chain is a kappa chain and the kappa
`variable regions show higher homology with the
`mouse sequences than a lambqa light variable
`""''~\
`(SbQ LD ND: w '1"~ IJ
`tSW'-CD NP'. IC?)
`region, e.g. KOL/\.(see below).
`RElAwas cbosen in
`preference to another kappa light chain because
`the X-ray structure of the light chain has been.
`determined so that a structural examination of
`individual residues could be made.
`12.1.2. HEAVY CHAIN
`Similarly Figure 4 shows an alignment of sequ~nces
`?i
`l5ffi :it t"\D :;
`{'SeQ Ii) l'UJ'.10)
`for the human framework region KOL arid the OKT3
`/\
`(\
`heavy variable region.
`The structural loops and
`CDRs believed to correspond to the antigen binding
`region are marked. Also marked are a number of
`other residues which may also contribute to
`antigen binding as described in 12.l(c).
`The
`residue type key and other indicators used in
`Figure 4 are ~he same as those used in Figure 3.
`(_)~'Q-;r,\) ~.1tJ)
`KOLAwas chosen as the heavy chain framework
`because the X-ray structure has been determined to
`a better resolution than, for example, NEWM and
`
`12.2.
`
`also the se~enc~ftlignment of OKT3 heavy variable
`'L~.n ~ 'NC)'
`~~ 4'J> '({?;If)~
`region~snowe a s ightly better homology to KO~( ~~
`' >
`than to NEWM.
`DESIGN OF VARIABLE GENES
`The variable region domains were designed with
`mouse variable region optimal codon usage
`[Grantham and Perrin (ref. 15)] and used the B72.3
`signal sequences [Whittle et al (ref. 13)].
`The
`sequences were designed to be attached to the
`constant region in the same way as for the ·
`chimeric genes described above.
`Some constructs
`contained the "Kozak consensus sequence" [Kozak
`(ref. 16)] directly linked to the 5' of the signal
`
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`

`12.3.
`
`- 40 -
`
`This sequence motif is
`sequence in the gene.
`believed to have a beneficial role in translation
`initiation in eukaryotes.
`GENE CONSTRUCTION
`To build the variable regions, various strategies
`are available.
`The sequence may be assembled by
`using oligonucleotides in a manner similar to
`Jones et al (ref. 17) or by simultaneously
`replacing all of the CDRs or loop regions by
`oligonucleotide directed site specific mutagenesis
`in a manner similar to Verhoeyen et al (ref. 2).
`Both strategies were used and a list of
`constructions is sCt out in Tables 1 and 2 and
`.
`a-
`d
`.
`in several cases
`Figures 4 and 5.
`It was note
`/\
`that the mutagenesis approach led to deletions and
`rearrangements in the gene being remodelled, while
`the success of the assembly approach was very
`sensitive to the quality of the oligonucleotides.
`
`13.
`
`CONSTRUCTION OF EXPRESSION VECTORS
`Genes were isolated from Ml3 or SP65 based
`intermediate vectors and cloned into pEE6hCMVneo
`for the light chains and pEE6hCMVgpt for the heavy
`chains in a manner similar to that for the
`chimeric genes as described above.
`
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`

`- 41 -
`
`TABLE 1
`CODE
`
`CDR-GRAFfED GENE CONSTRUCTS
`MOUSE SEQUENCE
`CONTENT
`
`METHOD OF
`CONSTRUCTION
`
`KOZAK
`SEQUENCE
`
`+
`
`ALL HUMAN FRAMEWORK REl
`
`LIGHT CHAIN
`121
`
`121A
`
`121B
`
`221
`
`221A
`
`221B
`
`221C
`
`26-32,
`26-32,
`
`50-56, 91-96 inclusive
`50-56, 91-96 inclusive
`46. 47
`+l, 3.
`50-56, 91-96 inclusive
`26-32,
`+ 46,
`47
`24-24,
`50-56, 91-96 inclusive
`24-34,
`50-56, 91-96 inclusive
`+l, 3,
`46, 47
`24-34,
`50·56, 91-96 inclusive
`+l, 3
`24-34,
`
`SDM and gene
`
`assembly
`
`Partial gene
`
`assembly
`
`n.d.
`+
`n.d. +
`
`Partial gene assembly
`
`n.d. +
`
`Partial gene assembly
`Partial gene assembly
`
`+
`
`+
`
`Partial gene assembly
`
`+
`
`Partial gene assembly
`
`+
`
`+
`+
`
`+
`
`+
`
`50-56, 91-96 inclusive
`
`HEAVY
`121
`
`131
`
`141
`321
`331
`
`CHAIN
`
`26-32,
`26-32,
`26-32,
`26-35,
`26-35,
`
`ALL HUMAN FRAME~ORK KOL
`50-56, 95-lOOB inclusive
`50-58, 95-lOOB inclusive
`50-65, 95-lOOB inclusive
`50-56, 95-lOOB inclusive
`50-58, 95-lOOB inclusive
`
`341
`
`26-35, 50-65, 95-lOOB inclusive
`
`Gene assembly
`Gene assembly
`Partial gene assembly
`Partial gene assembly
`Partial gene assembly
`Gene assembly
`SDM
`Partial gene assembly
`
`341A
`
`26-35, 50·65, 95-lOOB inclusive
`
`Gene assembly
`
`n.d. +
`n.d. +
`n.d.
`+
`n.d.
`
`+
`
`+
`
`+
`
`+
`
`+
`n.d.· +
`
`+6, 23, 24, 48, 49, 71, 73, 76,
`
`78~8.-8,n 91 (+63 -,human)
`(_'Z>V'-'< ~.Y NO:<\~U,
`26-35, 50-65, 95-lOOB inclusive
`+ 48, 49, 71, 73, 76, 78, 88, 91
`
`341B
`
`(+63 + human)
`
`Gene assembly
`
`n.d. +
`
`~
`n.d.
`SDM
`Gene assembly
`Partial gene
`assembly
`
`not done
`Site directed mutagenesis
`Variable region assembled entirely from oligonucleotides
`Variable region assembled by combination of restriction
`fragments either from other genes originally created by SDM
`and gene assembly or by oligonucleotide assembly of part of
`the variable region and reconstruction with restriction
`fragments from other genes originally created by SDM and gene
`assembly
`
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`

`14.
`14.1.
`
`- 42 -
`
`EXPRESSION OF CDR-GRAFTED GENES
`PRODUCTION OF ANTIBODY CONSISTING OF GRAFTED LIGHT
`(gL) CHAINS WITH MOUSE HEAVY (mH) OR CHIMERIC
`HEAVY (cH) CHAINS
`All gL chains, in association with mH or cH
`produced reasonable a.mounts of antibody.
`Insertion of the Kozak consensus sequence at a
`position 5' to the ATG (kgL constructs) however,
`led to a 2-5 fold improvement in net expression.
`Over an extended series of experiments expression
`levels were raised from approximately 200ng/ml to
`approximately 500 ng/ml for kgL/cH or kgL/mH
`combinations.
`When direct binding to antigen on HUT 78 cells was
`measured, a construct designed to include mouse
`sequence based on loop length (gL121) did not lead
`to active antibody in association with mH or cH.
`A construct designed to include mouse s~quence
`rsvn ~D NO : 2/0J_
`based on Kabat CDRs ( gL22 l ~'"demonstrated some weak
`binding in association with mH or cH.
`However,
`when framework residues 1, 3, 46, 47 were changed
`from the human to the murine OKTJ equivalents
`based on the arguments outlined in Section 12.l
`antigen binding was demonstrated when both o f the
`new constructs, which were termed 121A and 221A
`were co-expressed with cH. When the effects of
`these residues were examined in more detail, it
`appears that residues 1 and 3 are not major
`contri.Quting re.$J...dues as the product of the gL221B
`CSEG "J,'9 NO· .1.: I J
`geneAshows little detectable binding activity in
`association witq ~H.
`The light chain product of
`\ )1::-GI !:\') ND. Z$
`gL221~, in which ouse sequences are present at 46
`and 47, shows good binding activity in association
`with cH.
`
`I - -,
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`

`14.2
`
`- 43 -
`
`PRODUCTION OF ANTIBODY CONSISTING OF GRAFTED HEAVY
`(gH) CHAINS WIT~ MOUSE LIGHT {mL) OR CHIMERIC
`LIGHT (cL) CHAINS
`Expression of ~he gH genes proved to be more
`difficult to achieve than for gL.
`First,
`inclusion of the Kozak sequence appeared to have
`no marked effect on expression of gH genes.
`Expression appears to be slightly improved but not
`to the same degree as seen for the grafted light
`chain.
`Also, it p~oved difficult to demonstrate
`production of expected quantities of material when
`the loop choice {amino acid 26-32) for CDRl is
`used, e.g. gH121, 131, 141 and no conclusions can
`{.5fJ] 4'.'1) \\l,D.' Ii)
`be drawn about these constructs.
`Moreover, co~expression of the gH341 geneAwith cL
`or mL has been variable and has tended to produce
`lower amounts of antibody than the cH/cL or mH/mL
`)
`(seG Ib ND'. II
`,
`.
`.
`combinations.
`The al t..erat,t~s to gHl._4 lA to
`( l~ r.\) 'ND: I~) L '5.t <.>.:: +'-1) ~· lJ ) , I
`produce gH341~ana gH341B lead to improved levels
`/\
`!\
`of expression.
`This may be due either to a general increase in
`the fraction of mouse sequence in the variable
`region, or to the alteration at position 63 where
`the residue is returned to the human amino acid
`Valine (Val) from Phenylalanine (Phe) to avoid
`possible internal packing problems with the rest
`of the human framework.
`This arrangement also
`occurs in gH331 and gH321.
`When gH321 or gH331 were expressed in association
`with cL, antibody was produced but antibody
`('S\i:D. :Lt ~Oi ll)
`binding activity was not detected.
`When the more conservative gH341 geneAwas used
`antigen binding could be detected in association
`with cL or mL, but the activity was only
`marginally above the background level.
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`

`14.3
`
`- 44 -
`
`When further mouse residues were substituted based
`an the arguments in 12.1, antigen binding could be
`clearly demonstrated far the antibody produced
`when kgB341A and kgH341B were expressed in
`association with cL.
`PRODUCTION OF FULLY CDR-GRAFTED ANTIBODY
`The kgL221A gene was co-expressed with kgH341,
`kgH341A or kgH341B.
`For the combination
`kgH221A/kgH341 very little material was produced
`in a normal COS cell expression.
`For the combinations kgL221A/kgH341A or
`kgH221A/kgH341B amounts of antibody similar to
`gL/cH was produced.
`In several experiments no antigen binding activity
`could be detected with kgB221A/gH341 or
`kgH221A/kgH341 combinations, although expression
`levels were very low.
`Antigen binding was detected when kgL221A/kgH341A
`or kgH221A/kgH341B combinations were expressed.
`In the case of the antibody produced f ram the
`kgL221A/kgH341A combination the antigen binding
`was very similar to that of the chimeric antibody.
`
`An analysis of the above results is given below.
`
`15.
`
`DISCUSSION OF CDR-GRAFTING RESULTS
`In the design of the fully humanised antibody the
`aim was to transfer the minimum number of mouse
`amino acids that would confer antigen binding onto
`a human antibody framework.
`15 .1.
`LIGHT CHAIN
`15.1.1. EXTENT OF THE CDRs
`For the light chain the regions defining the loops
`known from structural studies of other antibodies
`to contain the antigen contacting residues, and
`
`/I / .
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`

`- 45 -
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`those hypervariable sequences defined by Kabat et
`al (refs. 4 and 5) as Complementarity Determining
`Regions (CDRs) are equivalent for CDR2.
`For CDRl
`the hypervariable region extends from residues
`:I
`24-34 inclusive while the structural loop extends
`(S~G. .t'D r{DS;
`from 26-32 inclusive.
`In the case of OKT3Athere
`is only one amino acid difference between the two
`options, at amino acid 24, where the mouse
`sequence is a serine and the human frtunework REl
`has glutamine.
`For CDR3 the loop extends from
`residues 91-96 inclusive while the Kabat
`;q
`hypervariability extends from residues 89-97
`inclusive.
`For OKT3 amino acids 89, 90 and 97. ND if-lrd9
`{SEQ 1'.-D
`/
`:,...
`are the same between OKT3 and REl (Fig. 3 ~·· When
`constructs based on the loop choice for CDRl
`(gL121) and the Kabat choice (gL221) were made and
`co-expressed with mH or cH no evidence for antigen
`binding activity could be found for gL121, but
`trace activity could be detected for the gL221,
`suggesting that a single extra mouse residue in
`the grafted variable region could have some
`detectable effect.
`Both gene constructs were
`reasonably well expressed in the transient
`expression system.
`15.1.2. FRAMEWORK RESIDUES
`The remaining framework residues were then further
`examined, in particular amino acids known from
`X-ray analysis of other antibodies to be close to
`the CDRs and also those amino acids which in OKT3
`showed differences from the consensus framework
`for the mouse subgroup (subgroup VI) to which OKT3
`shows most homology.
`Four positions 1, 3, 46 and
`47 were identified and their possible contribution
`was examined by substituting the mouse amino acid
`for the human amino acid at each position.
`Therefore gL221A (gL221 + DlQ, Q3V, L46R, L47W,
`
`I
`'
`
`/
`
`/
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`

`- 46 -
`
`see Figure 3 and Table 1) was made, cloned in
`EE6hCMVneo and co-expressed with cH (pJA144). The
`resultant antibody was well expressed and showed
`good bindipg activity. When the related genes
`l';if:O .l-'V NiY. '2.-lJl
`gL221BA(gL221 + DlQ, Q3V) and gL221C (gL221 +
`L46R, L47W) were made and similarly tested, while
`both genes produced antibody when co-expressed
`with cH, only the gL221C/cH combination showed
`good antigen binding. When the gL121A (gL121 +
`DlQ, Q3V, L46R, L47W) gene was made and
`co-expressed with cH, antibody was produced which
`also bound to antigen.
`15.2.
`HEAVY CHAIN
`15.2.1. EXTENT OF THE CDRs
`For the heavy chain the loop and hypervariability
`analyses agree only in CDR3.
`For CDRl the loop
`region extends from residues 26-32 inclusive
`whereas the Kabat CDR extends from residues 31-35
`For CDR2 the loop region is from
`inclusive.
`50-58 inclusive while the hypervariable region
`covers amino acids 50-65 inclusive.
`Therefore
`humanised heavy chains were constructed using the
`framework from antibody KOL and with various
`combinations of these CDR choices, including a
`shorter choice for CDR2 of 50-56 inclusive as
`there was some uncertainty as to the definition of
`the end point for the CDR2 loop around residues 56
`The genes were co-expressed with mL or cL
`to 58.
`initially.
`In the case of the gH genes with loop
`choices for CDRl e.g. gH121, gH131, gH141 very
`little antibody was produced in the culture
`supernatants.
`As no free light chain was
`detected it was presumed that the antibody was
`being made and assembled inside the cell but that
`the heavy chain was aberrant in some way, possibly
`incorrectly folded, and therefore the antibody was
`
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`

`- 47 -
`
`In some experiments
`being degraded internally.
`trace amounts of antibody could be detected in 355
`labelling studies.
`As no net antibody was produced, analysis of these
`constructs was not pursued further.
`When, however, a combination of the loop choice
`and the Kabat choice for CDRl was tested (mouse
`amino acids 26-35 inclusive) and in which residues
`31 (Ser to Arg), 33 (Ala to Thr), and 35 {Tyr to
`His) were changed from the human residues to the
`mouse residue and compared to the first series,
`antibody was produced for gH321, kgH331 and kgH341
`when co-expressed with cL.
`Expression was
`
`generally low and could not be markedly improved
`by the insertion of the Kozak consensus sequence
`5' to the ATG of the signal sequence of the gene,
`as distinct from the case of the gL genes where
`such insertion led to a 2-5 fold increase in net
`antibody production. However, only in the case
`of gH341/m.L or kgH341/cL could marginal antigen
`binding activity be demonstrated. When tgeu :I.~ ND~l~
`kgH341 gene was co-expressed with kgL221~ the net
`yield of antibody was too low to give a signal
`above the background level in the antigen binding
`assay.
`15.2.2. FRAMEWORK RESIDUES
`As in the case of the light chain the heavy chain
`frameworks were re-examined.
`Possibly because of
`the lower initial homology between the mouse and
`human heavy variable domains compared to the light
`chains, more amino acid positions proved to be of
`interest.
`Two genes kgH341A and kgH341B were
`constructed, with 11 or 8 human residues
`respectively substituted by mouse residues
`compared to gH341, and with the CDR2 residue 63
`returned to the human amino acid potentially to
`
`; .Y!
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`

`15.3
`
`- 48 -
`
`Both showed antigen
`improve domain packing.
`binding when combined with cL or kgL221A, the
`kgH341A gene with all 11 changes appearing to be
`the superior choice.
`INTERIM CONCLUSIONS
`It has been demonstrated, therefore, for OKT3 that
`to transfer antigen binding ability to the
`humanised antibody, mouse residues outside the CDR
`regions defined by the Kabat hypervariability or
`structural loop choices are required for both the
`light and heavy chains.
`Fewer extra residues are
`needed for the light chain, possibly due to the
`higher initial homology between the mouse and
`human kappa variable regions~
`Of the changes seven (1 and 3 from the light chain
`and 6, 23, 71, 73 and 76 from the heavy chain) are
`predicted from a knowledge of other antibody
`structures to be either partly exposed or on the
`antibody surface.
`It has been shown here that
`residues 1 and 3 in the light chain are not
`absolutely required to be the mouse sequence;
`for the heavy chain the gH341B heavy chain in
`combination with the 221A light chain generated
`only weak binding activity.
`Therefore the
`presence of the 6, 23 and 24 changes are important
`to maintain a binding affinity similar to that of
`the murine antibody.
`It was important,
`therefore, to further study the individual
`contribution of othe other 8 mouse residues of the
`kgH341A gene compared to kgH341.
`
`and
`
`16.
`
`FURTHER CDR-GRAFTING EXPERIMENTS
`Additional.CDR-grafted heavy chain genes were
`prepared substantially as described above. With
`reference to Table 2 the further heavy chain genes
`were based upon the gh341 (plasmid pJA178) and
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`516 of 1849
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`

`

`',~'
`~'O· 1
`
`- 49 -
`c_5~Q '[_\)
`gH34 lA (plasm~d pJA185 ~with either mouse OKT3 or
`human KOL residues at .6, 23, 24, 48, 49, 63, 71,
`73, 76, 78, BB and 91, as indicated.
`The CDR(cid:173)
`grafted light chain g~nes us~d in these furthef
`l$\';G. .YD Nll'.tf>) L<Ct:Q::C.!> -ND:24> J~ "J:V (IO: i'I
`(~0..:fD Ni7~
`experiments were gL22~, gL2211;f gL221B~and gL221St
`as described above.
`
`/ .
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`

`- 50 -
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`TA,BLE 2
`
`OKT3 HEAVY CHAIN CDR GRAFTS
`
`1.
`
`gH341 and derivatives
`
`6
`
`Q
`
`I
`RES NUM
`
`OKT3vh
`
`gH3'fl
`gH341A
`
`gH341E
`gH341*
`
`gH341*
`gH341D
`gH341*
`
`gH34_1C
`
`23
`
`24
`
`48
`
`49
`
`63
`
`71
`
`73
`
`76
`
`78
`
`88
`
`91
`
`K
`
`S
`
`A
`
`S
`
`A
`A
`
`A
`A
`
`I
`V
`I
`I
`I
`I
`I
`I
`V
`
`G
`
`A
`
`G
`G
`
`G
`G
`
`G
`G
`A
`
`G
`
`F
`F
`V
`V
`V
`V
`V
`V
`F
`
`T
`
`R
`
`T
`T
`
`T
`R
`T
`R
`R
`
`T
`
`K
`N
`K
`K
`
`K
`N
`K
`N
`N
`
`S
`N
`S
`S
`
`N
`N
`N
`N
`N
`
`S
`
`A
`
`L
`
`A
`A
`
`A
`A
`
`L
`L
`L
`
`A
`
`A
`
`G
`A
`G
`
`G
`G
`
`G
`G
`G
`
`A
`
`Y
`F JA178
`Y JA185
`G JA198~
`F JA207--=-
`F JA209
`F JA197
`F JA199
`F JA184
`Y JA203
`Y JA205
`Y JA183
`F JA204
`
`F JA206
`F JA208
`
`F
`
`K
`K
`K
`K
`
`K
`K
`
`N
`
`T
`
`T
`T
`
`T
`T
`
`R
`
`S
`
`S
`S
`
`S
`N
`
`N
`
`A
`
`A
`A
`
`A
`A
`
`L
`
`A
`
`A
`G
`
`G
`G
`
`G
`
`E
`Q
`K
`Q
`K
`9
`K
`g
`K
`K
`A
`Q
`Q
`A
`K
`_..Q ___ K __ A
`Q
`S
`E
`S
`E
`g
`E
`
`A
`A
`S
`A
`
`A
`A
`
`S
`S
`
`S
`S
`
`gH341*
`
`gH341*
`gH341B
`
`gH341*
`
`gH341.*
`
`gH341~
`
`I
`I
`I
`I
`
`I
`I
`
`V
`V
`V
`V
`
`V
`V
`
`G
`
`G
`G
`
`G
`G
`
`E
`
`KOL
`S
`?,O
`USf-'Q ·-r:;v NtO: ;;(, ID Gf\d l \- ii+)
`OKT3 LIGHT CHAIN CDR GRAFTS
`
`S
`
`V
`
`A
`
`2.
`
`gL221 and derivatives
`
`RES NUM
`
`1
`47
`46
`3
`g
`v
`R W
`Q
`L
`L DA221
`D
`...,g _ _...;v _ __;R~_.;.;.w 0A2 21A
`V
`L
`L DA221B
`Q
`Q
`R W DA221C
`Q iq L
`lo£Q :\_ \) ~CY. ,S\<D IC\ arr\ ~2Jb ---1%)
`
`OKT3vl
`
`GL221
`
`gL221A
`
`gL221B
`
`GL221C
`
`REl
`
`D
`
`D
`
`L
`
`MURINE RESIDUES ARE UNDERLINED
`
`,.
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`

`- 51 -
`
`The CDR-graf ted heavy and light chain genes were
`co-expressed in COS cells either with one another in
`various combinations but also with the corresponding
`murine and chimeric heavy and light chain genes
`substantially as described above.
`The resultant antibody
`products were then assayed in binding and blocking assays
`with EPB-ALL cells as described above.
`
`The results of the assays for various graft~d heavy ~ains
`rs \2'.'.Q ~D l"lu : z.
`co-expressed with the gL22lC light chai~are given i
`Figures 7 and 8 (for the JA184, JA185, JA197 and JA198
`constructs - see Table 2), in Figure 9 (for the JAl8~,
`tx iAria b
`JA184, JA185 and JA197 constructs) in Figure 10 (for the
`I\
`chimeric, JA185, JA199, JA204, JA205dnA'\A207, JA208 and
`JA209 constructs) and in Figure 11;(ror the JA183, JA184,
`JA185, JA198, JA203, JA205 and JA206 constructs).
`
`The basic grafted product without any human to murine
`~)
`.
`(2F..R J:D NQ:l.5;
`changes in the variable frameworks, i.e.)gL221/\
`Ls ro. :t.)) V'(!l • i •
`( JAl 78 k and also the "fully
`co-expressed with gh34 l
`grafted" product, having most human to murine changes in )
`CSEfJ :J-b NC·. 2.-~
`·
`·
`the grafted heavy chain framework, i.e. gL221CA
`(SfG U> ~0:12)
`co-expressed with gh341A (JA185~, were assayed for
`relative binding affinity in a competition assay against
`rnurine OKT3 reference standard, using HPB-ALL cells.
`The
`assay used was as described above in section 3.3.
`The
`results obtained are given in Figure 12 for the basic
`grafted product and in Figure 13 for the fully grafted
`product.
`These results indicate that the basic grafted
`product has neglibible binding ability as compared with
`the OKT3 murine reference standard; whereas the "fully
`grafted" product has a binding ability very similar to
`that of the OKT3 murine reference standard.
`
`The binding and blocking assay results indicate the
`following:
`
`.--·
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`- 52 -
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`The JA198 and JA207 constructs appear to have the best
`binding characteristics and similar binding abilities,
`both substantially the same as the chimeric and fully
`grafted gH341A products.
`This indicates that positions
`88 and 91 and position 76 are not highly critical for
`maintaining the OKT3 binding ability; whereas at least
`some of positions 6, 23, 24, 48, 49, 71, 73 and 78 are
`more important.
`
`This is borne out by the finding that the JA209 and JA199,
`although of similar binding ability to one another, are of
`lower binding ability than the JA198 and JA207
`constructs.
`This indicates the importance of having
`mouse residues at positions 71, 73 and 78, which are
`either completely or partially human in the JA199 and
`JA209 constructs respectively.
`
`Moreover, on comparing the results obtained for the JA205
`and JA183 constructs it is seen that there is a decrease
`in binding going from the JA205 to the JA183 constructs.
`This indicates the importance of retaining a mouse residue
`at position 23, the only position changed between JA205
`and JA183.
`
`These and other results lead us to the conclusion that of
`the 11 mouse framework residues used in the gH341A ·(JA185)
`construct, it is important to retain mouse residues at all
`of positions 6, 23, 24, 48 and 49, and possibly for
`maximum binding affinity at 71, 73 and 78.
`
`Similar Experiments were carried out to CDR-graft a number
`of the rodent antibodies including antibodies having
`specificity for CD4
`(OKT4), ICAM-1 (R6-5), TAG72 (B72.3),
`and TNF0(.(61E71, 101.4, hTNFl, hTNF2 and hTNF3).
`
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`EXAMPLE 2
`
`CDR-GRAFTING OF A MORINE ANTI-CD4 T CELL
`RECEPTOR ANTIBODY, OKT4A
`Anti ORT4A CDR-grafted heavy and light chain genes were
`prepared, expressed and tested substantially as described
`above in Example 1 for CDR-grafted OKT3.
`The CDR
`grafting of OKT4A is described in detail in Ortho patent
`application PCT/GB 90 .•..•.... of even date herewith
`entitled "Humanised Antibodies".
`The disclosure of this
`Ortho patent application PCT/GB 90 •..••.•.• is
`incorporated herein by reference.
`A number of
`CDR-grafted OKT4 antibodies have been prepared.
`Presently the ·CDR-grafted OKT4A of choice is the
`combination of the grafted light