W091109967
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`- 41 -
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`PCT/GB90/02017
`
`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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`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 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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`14. 2
`
`- 43 -
`
`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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`14.3
`
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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 Table 1) was made, cloned in
`EE6hCMVneo and co-expressed with cH (pJA144). The
`resultant antibody was wel l expressed a nd showed
`good bindi9q activity. When the rela ted genes
`l)fD .).."<;:!
`['ol~·.t..~l
`gL2 21BA( gL221 + Dl Q, Q3V) and gL221C (gL221 +
`L46R, L47W ) were made and similarly tested, whi l e
`both genes produced antibody when co-expressed
`with cH, only the gL221C/c.H combination s howed
`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.
`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 r e sidues 26-32 inclusive
`whereas the Kabat CDR e xtends from residues 31-35
`incl usive.
`For CDR2 the loop region is from
`S0-58 inclusive while the hypervariable region
`c overs amino acids 50-65 incl usive.
`Therefore
`humani sed heavy chains were constructed using the
`f ramework from antibody KOL and with various
`combinations of these CDR choices, including a
`shorter choice for CDR2 of S0-56 inclusive as
`t here 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
`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 t herefore the an tibody 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
`
`- 48 -
`
`Both showed antigen
`improve domain packing.
`binding when combined with cL or kgL221A, 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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`. (J--'
`~'0· 'l
`
`- 49 -
`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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`

`

`PCT / GB90/02017
`
`wo 91/09967
`
`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 residues
`are preferably donor murine amino acid residues in
`accordance with the present invention.
`A comparison of the amino acid sequences of the donor
`murine light chain variable domain and the RE1 human
`acceptor light chain variable further reveals that the
`murine and human residues are identical at all of
`positions 46, 48 and 71 and at all of positions 2, 4, 6,
`35, 36, 44, 47, 62, 64-69, 85, 87, 98, 99 and 101 and 102.
`However the amino acid residue at position 58 in LCDR2 is
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`the human REl framework residue not the mouse OKT4 residue
`as would be pref erred in accordance with the present
`invention.
`
`THE HEAVY CHAIN
`The human acceptor f ramework used for the grafted heavy
`(Sf.G. L-'t> w·. It)~
`.
`cha1.ns was KOL,.r
`The preferred CDR graft HCDR10 he avy chain has human to
`mous e changes at positions 24, 35, 57, 58, 60, 88 and 91
`in addition to the structural l oop CDRs.
`Of thes e positions , positions 35 (CDR1) and positions 57,
`58 and 60 (CDR2 ) fall within the pre f erred extended CDRs
`of the present invention. Also the human t o mouse change
`at position 24 corresponds to a posi tion at which the
`amino acid residue is a donor murine residue in accordance
`with the present invention. Moreover, the human to mouse
`changes at positions 88 and 91 correspond to positions at
`which the amino acid residues are optionally donor murine
`residues.
`Mor eover, a comparison o f the murine OKT4A and human KOL
`heavy chain variable amino acid sequences reveals that the
`murine and huma n res i dues are ide ntical at all of
`positions 2 3 , 49, 7 1, 73 and 78 and at all of positions 2,
`4, 6, 25 , 36, 37, 39, 47, 48, 93, 94, 103, 104, 106 and
`107.
`Thus the OKT4A CDR-grafted heavy chain HCDR10 corresponds
`t o a particularly preferred embodiment according to the
`present inve ntion •
`
`. /
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`EXAMPLE 3
`
`CDR-GRAFTING OF AN ANTI-MUCIN SPECIFIC MURINE
`ANTIBODY, 872. 3
`The cloning of the genes coding for the anti-mucin
`specific murine monoclonal antibody 872.3 and the
`preparation of B72.3 mouse-human chimeric antibodies has
`been described previously (ref. 13 and WO 89/01783).
`CDR-grafted versions of B72.3 were prepared as follows.
`(a)
`872.3 Light Chain
`CDR-grafting of this light chain was accomplished
`by direct transfer of the murine CDRs into the
`framework of the human light chain REl.
`The regions transferred were:
`
`CDR Number
`1
`2
`3
`
`Residues
`24-34
`50-56
`90-96
`
`The activity of the resulting grafted light chain
`was assessed by co-expression in COS cells, of
`genes for the combinations:
`B72.3 cB/B72.3 cL
`B72.3 cH/B72.3 gL
`
`and
`
`Supernatants were assayed for antibody
`concentration and for the ability to bind to
`microtitre plates coated with mucin.
`The
`results obtained indicated that, in combination
`with the 872.3 cH chain, 872.3 cL and 872.3 gL
`OJ'~ £1
`had similar binding properties.
`( SeQ 1:~ 00'·V
`Comparison of the murine 872.3 and REIAlight chain amino
`acid sequences reveals that the residues are identical at
`positions 46, 58 and 71 but are different at position 48 •
`
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`Thus changing the human residue to the donor mouse residue
`at position 48 ma y further improve t he b i nding
`c haracteristics of the CDR- grafted light chain, (872.3 gL }
`in accordance with the present invention.
`
`(b)
`
`872 . 3 heavy chain
`i .
`Choice of framework
`At the outset it was necessary to make a
`choice of human framework .
`Simply put,
`the question was as follows : Was it
`necessary to use the framework regions from
`an antibody whose crystal structure was
`known or could the choice be made on some
`other criteria?
`For 872.3 heavy chai n , it wa s reasoned
`that, while knowledge of structure was
`important, transfer of the CDRs from mouse
`to human frameworks might be facilitated if
`t he overall homology between the donor and
`receptor frameworks was maximised.
`Comparison of the B72.3 heavy chain
`sequence with those i n Kabat (ref. 4 ) for
`human heavy chains showed c lear{Jf.~n.t§ (UD'.!OJ
`872.3 had poor homology for KOLAand NEWM
`(for which crystal structures are
`available) but was very homologous to the
`heavy chain for EU.
`On this basis, EU was c hosen for the
`CDR-grafting and the following residues
`transferred as CDRs.
`
`CDR Number
`1
`2
`3
`
`Res idues
`27-36
`50-63
`93-102
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`Also it was not i ced that the FR4 region of
`EU was unlike that of any other human (or
`mouse ) antibody .
`Consequently , in the
`grafted heavy chain genes this was also ·
`changed to produce a "consensus" human
`sequence.
`(Preliminary experiments showed
`that grafted heavy c hain genes containing
`the EU FR4 sequence expressed very poorly
`in transient expression systems.)
`ii. Results with grafted heavy chain genes
`Expression of grafted heavy chain genes
`containing all human fr amework regions with
`either gL or cL genes produced a grafted
`antibody with little ability to bind to
`The grafted antibody had about 1%
`mucin.
`the activity of t he c himeric antibody.
`In these experiments, however, it was noted
`t hat the activity of the grafted antibody
`could be increased to,.vlO% of B72.3 by
`exposure to pBs o f 2 - 3 .5.
`This observation provi ded a clue as to how
`the activity of t he grafted antibody could
`be improved without ac i d treatment.
`It
`was p ostulated that acid exposure brought
`about the protonation of a n acidic residue
`(pKa of aspart ic acid • 3 .86 and of
`glutamine acid = 4.2 5) which in turn caused
`a change in structure of t he CDR loops , or
`allowed better acces s of antigen.
`From comparison of the sequences of B72.3
`(re f. 13) and EU ( refs. 4 and 5 ) , it was
`clear that, in going f r om the mouse to
`human frameworks, only two pos i t i o ns . had
`been changed in such a way that acidic
`These
`residues had been introduced.
`
`:' .
`
`..
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`/
`
`positions are at residues 73 and 81, where
`K to ~ and Q to E changes had been made,
`respec.tively.
`Which of these positions might be important
`was determined by examining the crystal
`structure of the KOL aniibody.
`In KOL
`(.5 t;:.~ '{::\) ~·. 10
`heavy chain, pos~tion 8
`is far removed
`.
`.A
`from e~ther of the CDR loops.
`Position 73, however 1 is close to both CDRs
`1 and 3 of the heavy chain and 1 in this
`position it was possible to envisage that a
`K to E change in this region could have a
`detrimental effect on antigen binding.
`iii. Framework changes in B72.3 gH gene
`On the basis of the above analysis 1 E73 was
`mutated to a lysine (K).
`It was found
`that this change had a dramatic effect on
`the ability of the grafted Ab to bind to
`mucin.
`Further the ability of the grafted
`B72.3 produced by the mutated gH/gL
`combination to bind to mucin was similar to
`that of the B72.3 chimeric antibody.
`iv. Other framework changes
`In the course of the above experiments,
`other changes were made in the heavy chain
`framework regions. Within the accuracy of
`the assays used 1 none of the changes,
`either alone or together, appeared
`beneficial.
`Other
`All assays used measured the ability of the
`grafted Ab to bind to mucin and, as a whole,
`indicated that the single framework change
`at position 73 is sufficient to generate an
`antibody with similar binding properties to
`B72.3.
`
`v.
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`Comparison of the B72.3 murine and EU he avy
`chain sequences reveals that the mouse and
`human residues are identical at positions
`23, 24, 71 and 78 .
`Thus the mutated CDR-grafted B72. 3 heavy
`chain corresponds to a preferred embodiment
`of the present invent i on.
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`EXAMPLE 4
`
`CDR-GRAFTING OF A MURINE ANTI- I CAM-1 MONOCLONAL ANTIBODY
`A murine antibody, R6- 5-D6 (EP 0314863 ) having specificity'
`for Intercellular Adhesion Molecule 1 (ICAM-1) was
`CDR-grafted substantially as described above in previous
`examples.
`This work is described in greater detail in
`co-pending application, British Patent Application No.
`9009549.8, the disclosure of which is incorporated herein
`by reference.
`The human EU framework was used as the acceptor framework
`for both heavy and light chains.
`The CDR-grafted
`antibody currently of choice i~_provided ~y co-expression
`l'S~ ~\) \\(.)~ 'l-Ie)
`of grafted liqht,chain gL221A and grafted heavy chain
`(.';)~ -n 1'{0·. ll? J
`;\
`gH341D~which has a binding affinity for !CAM 1 of about
`75% of that of the corresponding mouse-human chimeric
`antibody.
`LIGHT CHAIN
`gL221A has murine CORs at positions 24-34 (CDR1), S0-56
`(CDR2) and 89-97 (CDR3).
`In addition several framework
`residu_es are also the murine amino acid.
`These residues
`were chosen after consideration of the possible
`contribution of these residues to domain packing and
`stability of the conformation of the antigen binding
`region.
`The residues which have been retained as mouse
`are at positions 2, 3, 48 (?), 60, 84, 85 and 87.
`Comparison of the murine anti-ICAM 1 and human EU light
`chain amino acid sequences reveals that the murine and
`human residues are identical at positions 46, 58 and 71.
`HEAVY CHAIN
`gH341D has murine CDRs at positions 26-35 (CDR1), 50-56
`(COR2) and94-100B (CDR3).
`In addition murine residues
`were used in gH341D at positions 24, 48, 69, 71, 73i 80,
`88 and 91.
`Comparison of the murine anti-ICAM 1 and
`human EU heavy chain amino acid sequences are identical at
`positions 23, 49 and 78.
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`EXAMPLE 5
`
`CDR-Grafting of murine anti-TNFa an t ibodies
`A number of murine anti-TNFa monoclonal antibodies were '
`CDR- grafted substantially as descri bed above in previous
`examples.
`These antibodies include t he murine monoclonal
`A
`antibodies designated 61 E71, hTNFl, hTNF3 and 1 01 .4
`brief summary of the CDR-grafting of each of these
`antibodies is given below.
`
`61E71
`A similar analysis as described above (Example 1, Section
`12.1.) was done for 61E7l and for the heavy chain 10
`residues were identified at 23, 24, 48, 49, 68, 69, 71,
`73, 75 and 88 as residues to pote ntially retain as
`murine.
`The human frameworks chosen for CDR-grafting of
`t his antibody, and the hTNF3 and 101.4 antibodies were REl
`for the light chain and KOL for the heavy chain .
`Three genes were built , the first of which contained 23,
`24, 48, 49, 71 and 73 [gH341( 6) ] as murine residues.
`The
`second gene also had 75 and 88 as murine residues
`[gH341(8)] while the third gene additionally had 68, 69,
`Each was
`75 and 88 as murine residues [gH341(10 )] .
`co- expressed with gL221, the minimum grafted light chain
`(CDRs only).
`The gL221/gH341(6) and gL221/gH341(8)
`a ntibodies both bound