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`region of the variable r egion from the Aval site
`and to include the 5 ' r esidues of the human
`constant region up to and including a unique Narl
`site which had been previously engineered into the
`constant region.
`A Bindlll site was introduced to act as a marker
`for insertion of the linker.
`The linker was ligated to the VL fraqment and the
`413 bp EcoRl-Narl adapted fraqme nt was puri f ied
`from the ligation mixture.
`The constant region was isolated as an Narl-BamHl
`fragment from an Ml3 clone NW361 and was ligated
`with the variable region DNA into a n
`EcoRl / BamHl / ClP pSP65 treated vector in a three
`way reaction t o yield plasmid J A143.
`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-~A~s~n_-~Ar~g~/ ____ -~/~T~h=r-val-Ala
`VARIABLE
`CONSTANT
`··-·- This arrangement of sequence introduces a
`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 f rom the mouse constant
`region, Alanine (Ala).
`
`:rYS>'>)
`L$Q
`-Ala /1
`
`9.2
`
`, --·-···- -,--·- ·-
`
`,-, ,
`
`I
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`i nc l uded i n this
`An internal Hindlll site was not
`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 ligated to Narl cut pNW361 and then the
`adapted 396bp constant region was isolated af ter
`recutting the modified pNW3 61 with EcoRl.
`The
`variable region fragment and the modified constant
`region fragment were ligated directly into
`EcoR l /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 t he insert in the correct orientation were
`obtained and the adapter sequence of one was
`confirmed by DNA sequenc i ng
`HRAVY CHAIN GENE CONSTRUCTION
`9.3.
`9 .3. 1 . CHOICE OF HEAVY CHAIN GENE ISOTYPE
`The constant region isotype c hos en for the heavy
`chain was human IgG4.
`9.3.2. GENE CONSTRUCTION
`The heavy chain eDNA sequence showed a Banl site(seQ r::p I'JO'.o)
`near the 3' end of the variable region (Fig. 2(a)J.
`)1
`The majority of the sequence of the variable
`region was isolated as a 4 26bp. EcoRl/ClP/Banl
`fragment.
`An oligonucleotide adapter was
`designated to replace the remainder of the 3'
`region of the variable region from the Banl site
`up to and including a unique Hindiii site which
`had been previ ously engineered into t he first two
`amino a cids of the constant regio n.
`The linker was ligated to the VB fragment and the
`EcoRl-Hindlll adapted fragment was purified from
`the ligation mixture.
`
`\
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`The variable r egion was ligated to the constant
`region by c utting pJA91 with EcoRl and Hindlll
`removing the intron fragment and replacing it with
`the VH to yield pJA142.
`Clones were isolated
`after transformation i nto E.coli JM101 and the
`linker and j unction sequences were confirmed by
`DNA sequencing.
`(N.B . The Hindlll site is lost
`on cloning) .
`
`CONSTRUCTION OF CHIMERI C EXPRESSION VECTORS
`neo AND gpt VECTORS
`The chimeric light chain {vers i on l) was removed
`from pJA14 3 a s an EcoRl fragment and cloned into
`EcoRl/ClP treated pEE6hCMVneo expression vector to
`yield pJA145.
`Clones with the insert in t he
`correct orientation were identified by restrict i on
`mapping.
`The chimeric light chain (version 2 ) was
`constructed a s described above.
`The chimeric heavy chain gene was isolated f r om
`pJA142 as a 2 .5Kbp EcoRl / BamHl f ragment and cloned
`into the EcoRl / Bcl l/ClP treated vector fragment of
`a derivative of pEE6hCMVgpt to yield plasmid
`pJA144.
`GS SEPARATE VECTORS
`GS versions of pJA14 1 and pJA144 were constructed
`by replacing the neo and gpt cassettes by a
`BamHl / Sall / ClP treatment o f the plasmids,
`isolation of the vector fragment and l igation 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
`(chi meric l i ght), 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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`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 BamB1/C1P and ligating in a Bgl11/Hindl11
`hCMV promoter cassette along with either the
`Hind111/BamB1 fragment from pJA141 into pJA180 to
`give the cH-cL-GS plasmid pJA182 or the
`Hind111/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 pJA14S (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
`In this case the light chain did not
`expressed.
`show an aberrant mobility or a size shift in the
`presence or absence of tunicamycin.
`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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`12.
`
`12. 1.
`
`- 37 -
`
`EXPRESSION IN CHINESE HAMSTER OVARY (CHO) CELLS
`Stable cell lines have been prepared from plasmids
`PJA141 / pJAl44 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 ant igen 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 posit ioning the c ontact
`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:
`(a)
`By examination of antibody X-ray crystal
`structures the antigen binding surface can
`be predominantly located on a series of
`loops, three per domain, which extend from
`the B-barrel framework.
`By analys i s 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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`(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 • 1 . 1 . LIGHT CHA-GJb 4J;l NO~{b And "')
`Figure 3~shows an alignm1~~~~<ae~~ces for the
`human framework regio~~l and the OKTJ light
`l56:JW f\.Q'~t:
`It
`variable region.
`The
`ructural loops (LOOP) and
`A
`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 13.1~- ~J)
`(SE:Q J:O NQ:
`1 (Jr0
`'}
`Above the sequence in Figure 3A~he res~
`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
`Surface
`Exposed
`I -
`Interface
`Interface
`Packing/ Part Exposed
`? - Non-CDR Residues which may require to be left
`as Mouse sequence.
`
`E
`*
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`This sequence motif is
`sequence in the gene.
`believed to have a beneficial role in tran slation
`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
`con structions is set out in Tables 1 and 2 and
`a-{-
`Figures 4 and 5.
`It was noted in several cases
`1\
`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 Ml 3 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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`TABLE 1
`CODE
`
`CDR-GRAFTED GENE CONSTRUCTS
`MOUSE SEQUENCE
`CONTENT
`
`METHOD OF
`CONSTRUCTION
`
`KOZAK
`SEQUENCE
`
`+
`
`121B
`
`221
`221A
`
`LIGHT CHAIN
`ALL HUMAN FRAMEWORK REl
`121
`26-32, 50-56. 91-96 inclusive
`l21A
`26-32 , 50-56, 91-96 inclusive
`+1, 3. 46. 47
`26-32, 50-56, 91 - 96 inclusive
`+ 46, 47
`24 -24 , 50-56, 91-96 inclusive
`24-34 , 50-56, 91- 96 inclusive
`+1, 3, 46, 47
`24-34, 50- 56 , 91 - 96 inclusive
`+1. 3
`24-34, 50-56, 91-96 inclusive
`
`221B
`
`221C
`
`HEAVY CHAIN
`ALL HUMAN FRAMEWORK KOL
`121
`26-32, 50·56, 95 -lOOB inclusive
`131
`26- 32, 50-58, 95- l OOB inclusive
`141
`26 - 32, 50·65, 95-lOOB inclusive
`321
`26- 35, 50·56 , 95- l OOB inclusive
`331
`26-35, 50-58, 95-lOOB inclusive
`
`341
`
`26-35 , 50· 65, 95-lOOB inclusive
`
`341A
`
`341B
`
`26-35 , 50-65, 95-lOOB inclusive
`+6, 23 , 24, 48 , 49, 71, 73, 76,
`
`(~8~8~~ 9~\+-t:-u--ruman)
`26-35, 50-65 , 95- l OOB inclusive
`+ 48 , 49, 71, 73, 76, 78, 88, 91
`(+63 + human)
`
`SDM and gene
`Partial gene
`
`assembly
`assembly
`
`n . d.
`+
`n.d. +
`
`Parcial gene assembly
`
`n.d. +
`
`Parcial gene assembly
`Parci al gene assembly
`
`Parcial gene assembly
`
`+
`
`+
`
`+
`
`Par tial gene assembly
`
`+
`
`+
`+
`
`+
`
`+
`
`Gene assembly
`Gene assembly
`Partial gene assembly
`Parcial gene assemb l y
`Partial gene assembly
`Gene assembly
`SOH
`Par tial gene assembly
`Gene assembly
`
`n.d . +
`n . d. +
`n.d.
`+
`n. d .
`+
`+
`
`+
`
`+
`
`+
`n.d .· +
`
`Gene as sembly
`
`n.d. +
`
`KEY
`n .d .
`SDM
`Gene assembly
`Parcial gene
`assembly
`
`not done
`Site directed mutagenesis
`Variabl e region assembled entirely f r om oligonucl eotides
`Variabl e region assembled by combination of restriction
`fragments either from ocher genes ori ginally created by SDM
`and gene assembly or by ol i gonucleotide assembly of pare of
`the variable region and reconstruction with restriction
`fragments from other genes originally created by SDM and gene
`assembly
`
`-- ---
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`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 amounts 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 mB or cH.
`A construct designed to include mouse s~quence
`r .::.o r:o NO·.?.D_
`based on Kabat CDRs ( gL221 ~'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 OKT3 equivalents
`based on the arguments outlined in Section 12.1
`antigen binding was demonstrated when both of 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
`contr~uting r7ftdues as the product of the gL221B
`cs h -:til w:.t,_ 1 d
`bl b · d ·
`·
`· t
`·
`geneAs ows li t e etecta e ~n ~ng act~v~ y ~n
`association wit~ ~H.
`The l ight chain product of
`~~t:-U ~\) ND.Z$
`gL221~, in wbich ouse sequences are present at 46
`and 47, shows good binding activity in association
`with cH.
`
`I -·~
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`PRODUCTION OF ANTIBODY CONSISTING OF GRAFTED HEAVY
`( gH) CHAINS WIT~ MOUSE LIGHT
`( rnL ) OR CHIMERI C
`LIGHT (cL ) CHAINS
`Expression of .the gH genes proved to be more
`difficult to achieve than f o r gL.
`First,
`inclusion of the Kozak sequence appeared to have
`no marked effect on expressio n of gH genes.
`Expression appears to be slightly improved but not
`to the same degree as seen f o r the grafted light
`chain.
`Also, it p~oved difficult to demonstrate
`production of expected quantities of material when
`the loop choice (amino acid 26-3 2) f or CDRl is
`used, e.g. 9H121, 131, 141 and . no conclusions can
`~ :r)) NO: 11)
`be drawn about these constructs.
`Moreover, co~expressio n of the gH341 gene~ with cL
`o r mL has been variable and has tended to produce
`l ower amounts of antibody than the cH/cL o r mH/mL
`)
`( $6Q I.b NO'. I I
`.
`,
`.
`comb1.nat1.ons .
`The al t..erat,Lo.ns to gH~ l.A t o
`f"~-s;:,>~:l'l;-1 L~J:(,.(, ~.() \'IU5<2.J ) ....
`produce gH34l~ana gH341B lead t o ~mproved level s
`1\
`A
`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 prob l ems with the rest
`of the human framework.
`This arrangement also
`occurs in gH331 and gH32 1.
`When gH321 or gH331 were expr e s sed i n association
`with cL, ant i body was produced but a n tib ody
`( '5\!0- ~ ~Oi !1)
`binding act i vity was not detected.
`When the more c onservative gB34 1 geneAwas u~ed
`antigen binding could be detected i n association
`with cL or rnL, but the activity was only
`marginally above the background level.
`
`f
`
`•
`
`'
`•
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`When f urther mouse residues were substituted based
`on the arguments in 12.1, antigen binding could be
`clearly demonstrated for the antibody produced
`when kgB341A and kgB341B were expresse d in
`association with cL.
`PRODUCTION OF FULLY CDR-GRAFTED ANTIBODY
`The kgL221A gene was co-expressed wit h kgH341,
`kgH341A or kgH341B.
`For the combination
`kgH221A/ kgH341 very lit t l e mat erial was produced
`in a normal COS c e l l expression.
`For the combinations kgL221A/ kgH341A or
`kgH221A/ kgH341B amounts of antibody s imil ar t o
`gL/ cH was produced.
`In several experiments no antigen binding activi t y
`coul d be detected with kgB221A/ gH341 or
`kgH221A/ kgH341 combinations , a l though expression
`levels were very low.
`Ant igen binding was de t ec t ed when kgL221A/ kgH341A
`or kgH221A/ kgH341B combinations were expressed.
`In the case of the antibody produced from the
`kgL221A/kgH341A combination the antigen binding
`was very similar to that of the chimeric antibody.
`
`An analysis of the above results is given bel ow.
`
`15.
`
`DISCUSSION OF CDR-GRAFTING RESULTS
`I n the design of the ful l y humanised antibody the
`aim was t o transfer the minimum number of mouse
`amino acids that would confer ant igen binding onto
`a human antibody framework.
`LIGHT CHAIN
`15 . 1.
`15.1.1. EXTENT OF THE CDRs
`For the l ight c hain the regions defining the loops
`known from structural studies of other antibodies
`to contain t he antigen cont acting residues, and
`
`f • /
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`those hypervariable sequences defined by Kabat ~
`al (refs. 4 and 5 ) as Complementarity Determining
`Regions ( CDRs) are equivalent for CDR2 .
`For CDR!
`the hypervariable region extends from residues
`:1
`24-34 inclusive while the structural l oop extends
`(.)~Q .I'D Y'«)S;
`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 framework 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 0KT3 amino acids 89, 90 and 97 . N" 'b~rJ9
`{SEQ t:-D
`v_:,..
`/
`are the same between OKT3 and REl (Fig. 3~; When
`constructs based on the l oop choice for CDRl
`{gL12l) 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 an t ibodies 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 0KT3
`shows most homo logy.
`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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`see Figure 3 and Ta bl e 1) was made, cloned in
`EE6hCMVneo and co-expressed with cH (pJA144). The
`resultant antibody was wel l expressed and showed
`good bindi9q activity . When the related genes
`l)fD .).."<;:!
`['ol~·.t..~l
`gL221BA( gL221 + DlQ, Q3V) and gL221C (gL221 +
`L46R, L47W) were made and similarly tested, while
`bot h genes produced antibody when co-expressed
`with cH, only the gL221C / c.H combination showed
`good antigen binding. When the gL121A (gL121 +
`Dl Q, Q3V, L46R, L47W) gene was made and
`c o-expressed with cH, antibody was produced which
`also bound to antigen.
`HEAVY CHAIN
`15.2.
`15. 2.1. EXTENT OF THE CDRs
`For the heavy chain the loop and hypervariability
`analyses agree only in CDR3 .
`For CDRl t he loop
`region extends from residues 26-32 inclusive
`whereas the Kabat CDR e xtends from residues 31-35
`inclusive.
`For CDR2 the loop regi on is from
`S0- 58 inclusive while the hypervariable r e gion
`c overs amino acids 50-65 incl usive.
`Therefore
`humanised heavy chains were construc ted us ing the
`framework from antibody KOL and with various
`c ombinations of these CDR choices, including a
`shorter choice for CDR2 of S0-56 inclusive as
`there was some uncertainty as to the definition of
`the end point for the CDR2 loop around residues 56
`to 58 .
`The genes were co-expressed with mL or cL
`initially.
`In the case of the gH genes with loop
`choices for CDRl e.g. gH121, gH131, gB141 very
`little antibody was produced in the culture
`s upernatants.
`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 t herefore the antibody was
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`In some experiments
`being degraded internally.
`trace amoun~s of antibody could be detected in 35s
`labelling studies.
`As no net antibody was produced, analysis of t hese
`constructs was not pursued further.
`When, however, a combination of the loop choice -
`and the Kabat choice for CDRl was t e sted (mouse
`amino acids 26-35 i nc lusive) and in which residues
`31 (Ser to Arg ) , 33 {Ala to Thr ) , and 35 (Tyr to
`His) were changed f rom the human residues t o the
`mouse residue and c ompared to the first series,
`antibody was produced f or gH321, kgH33 1 and kgH341
`Expression was
`when co-expressed wi th cL.
`generally low and could not be markedly improved
`by the insertion of the Kozak consensus sequence
`5 ' to t he ATG of the s i gnal sequence of t he gene,
`as distinct from the case of the gL genes where
`such insert ion led to a 2-5 fold increase in net
`However, only in the case
`antibody production.
`of gH341 / mL or kgH341/cL could marginal antigen
`binding activity be demonstrated. When tg~ 'J.."\:1 1-JD'. 2. ~
`kgH341 gene was co-expressed with kgL221~ the net
`yield of antibody was t oo 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
`respecti vel y substitut ed by mouse residues
`compared to gH341, and with the CDR2 residue 63
`returned to the human amino acid potentially to
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`1 5. 3
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`Both showed antigen
`improve domain packing.
`binding when combined with cL or kgL2 21A , the
`kgH3 4 1A gene with all 11 changes appearing to be
`the superior choice.
`INTERIM CONCLUSIONS
`It has been demonstrated, therefore, for 0KT3 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 e xtra residues are
`needed for the light chain, possibly due to the
`higher initial homology between the mouse and
`human kappa variab le regions~
`Of the . changes seven (1 a nd 3 from the light chain
`and 6 , 23, 71 , 73 and 76 from the heavy chain ) are
`predicted from a know l edge of other antibody
`structures t o be eit her partly exposed or on t he
`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 c hain 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 pJAl 78) and
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`~'0· 'l
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`c.:;8Q '(_\)
`gH341A (pl asm~d pJA185 ~ wi th either mouse OKT3 or
`human KOL res~dues at _6 , 23 , 24 , 48, 4 9, 63, 71,
`The CDR(cid:173)
`73, 76, 78, 88 and 91, as indicate d.
`grafted light c hain g~nes us~d in these furthef
`(~:fDI'JV.i
`lSEQl>PNO:tb ) l~E:Q:COND:Zt> )~'l"J>!IO: Z!7
`experiments were gL22~ , gL221~ gL22 1BAand gL221~
`as described above.
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`TA@LE 2
`
`OKT3 HEAVY CHAI N CDR GRAFTS
`
`l.
`
`gH34l and derivatives
`
`- 50 -
`
`24 . 48 . 49
`A
`G
`I
`v
`s
`I
`A
`I
`I
`I
`I
`
`23
`K
`s
`K
`
`K
`
`K
`
`K
`
`K
`
`RES 1NUM
`OKT3vh
`gHJ4l
`gH341A
`gH34lE
`gH34l*
`gHJ41*
`gH341D
`gH341*
`gH34_1C
`gHJ4 l *
`gH341*
`gH34l.B
`gH341*
`gH34l*
`
`A
`
`A
`
`A
`
`A
`
`I
`v
`I
`I
`I
`I
`
`A
`A
`A
`
`A
`s
`
`A
`A
`
`gH341"*
`KOL
`(_pf-'Q ·t:-v
`OKTJ LIGHT CHAIN CDR GRAITS
`
`~6
`
`6
`g
`E
`g
`g
`g
`g
`g
`g
`K
`g
`K
`g
`s
`s
`E
`s
`E
`g
`s
`s
`E
`I
`g
`s
`I
`A
`v
`s
`s
`E
`f'.lO: ?f: ID Oild l! - t+)
`';0
`
`63
`
`F
`
`F
`v
`v
`v
`v
`v
`v
`F
`v
`v
`v
`v
`v
`v
`
`7l
`T
`R
`T
`T
`T
`R
`T
`R
`
`R
`T
`T
`
`T
`T
`T
`T
`R
`
`73
`K
`
`N
`
`K
`
`K
`
`K
`
`N
`
`K
`
`N
`
`N
`
`K
`
`K
`
`K
`K
`
`K
`K
`
`N
`
`76
`s
`N
`s
`s
`N
`N
`
`N
`
`N
`N
`s
`s
`s
`s
`s
`N
`
`N
`
`78
`
`A
`L
`
`A
`
`A
`
`A
`A
`L
`
`L
`
`L
`
`A
`
`A
`A
`
`A
`
`A
`
`A
`L
`
`88
`A
`G
`
`A
`G
`
`G
`G
`
`G
`
`G
`G
`A
`
`A
`
`A
`
`G
`G
`G
`G
`
`91
`y
`
`F JA178
`Y JA185
`G JA198-..._
`F JA207-=-
`F JA209
`F JA197
`F JA199
`F JA184
`Y JA203
`Y JA205
`Y J A183
`F JA204
`F J A206
`F JA208
`F
`
`A
`G
`G
`
`G
`
`G
`
`G
`
`G
`A
`G
`
`G
`
`G
`
`G
`
`G
`G
`
`A
`
`2.
`
`gL221 and derivatives
`
`RES ~ l
`g
`OKT3vl
`GL221
`D
`g
`gL2 21A
`g
`gU21B
`GL2 21C
`D
`REl
`
`D
`
`46
`
`47
`
`3
`v
`R
`\.l
`L DA221
`L
`Q
`v
`R W 0A221A
`v
`L
`L DA221B
`t.J DA221C
`R
`Q
`Q !).q L
`L
`
`MURINE RESIDUES ARE UNDERLINED
`
`~ ~ lo£Q ~\) ~o·. %\~ l q &fe\ 'ltD,.. n)
`
`------~
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`The CDR- grafted heavy and light chain genes were
`co-expressed in COS cells e i ther wit h 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 BPB-ALL cells as described above.
`
`The results of the assays for various graftgd heavy ~ains
`_f'i:> tc.Q 4=D NU : t~/
`co-expressed with the gL22lC light chai ,~are given in
`Figures 7 and 8 (for the JA184, JA185, JA197 and JA198
`constructs - see Table 2), in Figure 9 (for the JALB~,
`CJ. ~na b
`JA184, JA185 and JA197 constructs) i n Figure 10 (for the
`II
`chimeric, JA185, JA199, JA204, JA2051.~\A207, J A208 and
`JA209 constructs) and in Figure ll;(for the JA183, JA184,
`JA185, JAl98, JA203, JA205 and J A206 co nstructs).
`
`.
`The basic grafted product without any human to murine
`~J
`221(2F..R 4--D NQ.W;
`·
`·
`k
`·
`
`h c anges 1.n the varl.able framewor s, 1..e. ) gL
`.11
`[sro :tP IXb • \ I
`/ '
`co-expressed with gh341 (JA1 78~, and also the "fully
`grafted" product, having most human to murine changes in )
`tsEO :1-0 N.IY. 2..8
`.
`the grafted heavy chain framework , 1..e. gL22lCA
`cswn ~0: 1'2.)
`co-expressed with gh341A (JAlBS~, were assayed for
`relative binding affinity in a competition assay against
`murine 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 abil ity 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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`The JA198 and JA207 constructs appear to have the best
`binding characteristics and similar binding abilit ies,
`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 JA20 9 and JA199,
`alt hough of similar binding ability to one anot her, are of
`lower binding ability than the JA198 and JA207
`const ructs.
`This indicates t he import ance of having
`mouse residues at positions 71, 73 and 78, which are
`either completely or parti a l ly 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 JA2 05 to the JA183 const ructs.
`This indicates the import ance of retaining a mouse residue
`at position 23, the only position changed between JA205
`and JA163.
`
`These and other r e sults lead us t o the conclusion that of
`the 11 mouse framework residues used in the gH341A (JA185 )
`construct, it is import ant to retain mouse residues at all
`of positions 6, 23, 24, 48 and 49, and possibly for
`maximum binding affinit y a t 71, 73 and 76.
`
`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 TNF~( 6 1E71, 101.4, hTNFl, hTNF2 and hTNF3).
`
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`
`EXAMPLE 2
`
`CDR-GRAFTING OF A MURINE ANTI-CD4 T CELL
`RECEPTOR ANTIBODY, OKT4A
`Anti OKT4A 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 "Bwnanised 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-graf ted OKT4A of choice is the
`combination of the grafted light chain LCDR2 and the
`grafted heavy chain HCDR10.
`
`THE LIGHT CHAIN
`The human acceptor fr~e~o[k used for the grafted light
`.
`0'S p ~: ~o..ro ~
`chains was REJ,\-
`The pref rred LCDR2 light chain has
`human to mouse changes at positions 33, 34, 38, 49 and 89
`in addition to the structural loop CDRs.
`Of these
`changed positions, positions 33, 34 and 89 fall within the
`preferred extended CDRs of the present invention
`(positions 33 and 34 in CDRl and position 89 in CDR3).
`The human to murine changes at positions 38 and 49
`corresponds to positions at which the amino acid residu