Molecular Immunology, Vol. 28, No. 4/5, pp. 489—498, 1991
`Printed in Great Britain,
`
`0161-5890/91 $3.00 + 000
`Pergamon Press pic
`
`A POSSIBLE PROCEDURE FOR
`
`REDUCING THE IMMUNOGENICITY OF ANTIBODY
`
`VARIABLE DOMAINS WHILE PRESERVING THEIR
`
`LIGAND-BINDING PROPERTIES
`
`Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases,
`National Institutes of Health, Bethesda, MD 20892, U.S.A.
`
`EDUARDO A. PADLAN
`
`(First received 24 May 1990; accepted in revised form 17 September 1990)
`
`Abstract—It is proposed to reduce the immunogenicity of allogeneic antibody variable domains, while
`preserving ligand-binding properties, by reducing their antigenicity through replacement of the exposed
`residues in the framework regions which differ from those usually found in host antibodies. The results
`of a comparison of representative murine antibody sequences with those of human origin suggest that the
`number of residues that need to be replaced to “humanize” those antibodies could be small.
`
`INTRODUCTION
`
`Antibodies of predetermined specificity have many
`potential uses in therapy and diagnosis and hybrid-
`oma technology (Koehler and Milstein, 1975) has
`made possible the generation of virtually limitless
`amounts of such antibodies. Unfortunately, hybrid-
`oma proteins are more easily obtained from rodent
`sources and the use of those antibodies in human
`
`subjects will be hindered by the patients’ immune
`system. Various attempts have been made to make
`such allogeneic antibodies less immunogenic while
`preserving their antigenvbinding properties.
`The immunogenicity of rodent-derived antibodies
`had been reduced by generating chimeric antibodies
`in which the variable domains of the rodent proteins
`were grafted onto constant regions of human mol-
`ecules (reviewed by Morrison and Oi, 1988). Such
`chimeric molecules would be expected to possess all
`the antigen-binding characteristics of the original
`antibodies, but would retain the immunogenicity of
`the rodent variable domains.
`
`reduction in the immunogenicity of
`Further
`chimeric antibodies had been achieved by Winter
`and coworkers (Jones et al., 1986; Verhoeyen et (21.,
`1988; Riechmann et al., 1988) by grafting the comple-
`mentarity-determining regions, or CDRs, of the allo-
`geneic proteins onto human framework regions. In
`two published applications of this method (Riechmann
`et al., 1988; Queen at at, 1989), some of the frame.
`work residues of the allogeneic antibody, which were
`thought would ensure a pr0per structure for the
`
`
`Abbreviations: CDR, complementarity-determining region;
`Fab, the antigen-binding fragment of an antibody; Fv,
`the module containing VH and VL, the variable domains
`of the heavy and light chains, respectively.
`
`variable domains and a proper interdomain contact,
`were kept in the “humanized” molecule in an attempt
`to preserve ligand-binding properties. The “human-
`ized” molecules did exhibit the same ligand—binding
`specificity as the original antibodies, but with only
`about one-third of the affinity.
`X-ray crystallographic studies have repeatedly
`demonstrated that the framework structures of the Fvs
`of different antibodies assume a “canonica ” structure
`
`regardless of species of origin, amino acid sequence,
`or ligand—binding specificity. This is generally taken
`as evidence that the ligand-binding characteristics of
`an antibody combining site are determined primarily
`by the structure and relative disposition of the CDRs,
`although some neighboring framework residues also
`have been found to be involved in antigen binding
`(Amit et al., 1986; Colman et al., 1987; Sheriff et (11.,
`1987; Padlan et al., 1989). Thus, if the fine specificity
`of an antibody is to be preserved, its CDR structures
`(and probably also some of the neighboring residues),
`their interaction with each other, and their interaction
`with the rest of the variable domains must be strictly
`maintained. This may require the retention of most,
`if not all, of the many interior and interdomain
`contact residues; the structural effects of replacing
`only a few of them cannot be predicted.
`It may be possible to simultaneously reduce
`immunogenicity and strictly preserve ligand-binding
`properties. Since the antigenicity of a protein is de-
`pendent on the nature of its surface (Benjamin er £21.,
`1984), replacing the exposed residues of an allogeneic
`antibody with those usually found in host antibodies
`would decrease the probability of its recognition and
`internalization by antigen-processing cells, specifically
`by antigen-specific B-cells which play a critical role in
`priming T-cells (Ron et all, 1983; Kurtdones et at,
`1988; see also the review by Vitetta er al., 1989). Such
`
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`490
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`E. A. PADLAN
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`a procedure, in the end, then serves to reduce immuno-
`genicity. The judicious replacement of exterior residues
`should have little, or no, effect on the interior of
`the domains, or on the interdomain contacts. If the
`envisioned replacements are confined to the frame-
`work regions, especially to only the exposed residues,
`then the ligand-binding properties of the antibody
`could be expected to remain unaltered.
`This possibility is explored in this paper using as
`an example the “humanization” of mouse variable
`domains. First, the available sequence data for the
`various human variable domain subgroups are
`analysed in terms of the residues which usually occur
`at each framework position. Then the differences
`between human and representative mouse sequences
`are assessed and correlated with solvent accessibility
`as deduced from the known three-dimensional struc-
`ture of human and mouse Fabs. It is found that the
`
`number of exposed residues, which need to be replaced
`to “humanize” mouse variable domains, could be
`small.
`
`MATERIALS AND METHODS
`
`(a) Structural data
`
`The structures that were chosen for the analysis
`were those of the variable domains of the antibodies
`KOL and J539. The Fabs of KOL and .1539 are those
`
`which have been elucidated to the highest resolution
`and have been subjected to the most extensive refine—
`ments so far. The crystal structure of the human KOL
`Fab had been determined at 1.9 Angstrom resolution
`(Marquart et al., 1980) and had been refined to an
`R-value of 1.89; the structure of the mouse 1539 Fab
`had been elucidated at 1.95 Angstrom resolution and
`had been refined to an R-value of 1.95 (T. N. Bhat,
`E. A. Padlan and D. R. Davies, in preparation). The
`atomic coordinates for the KOL and .1539 structures
`are available from the Protein Data Bank (Bernstein
`et al., 1977) (File 2FB4 for KOL, File 2FBJ for J539).
`The solvent accessibility of the individual residues
`in the KOL and J539 VL and VH was assessed using
`program MS of Connolly (1983) and programs devel-
`oped by Sherifi" (Sheriff et al., 1985). A radius of L7
`Angstroms was assumed for the solvent probe; stan-
`dard van der Waals’ radii (Case and Karplus, 1979)
`were used. The fractional accessibility values for the
`sidechains (Sheriff et al., 1985; Shrake and Rupley,
`1973) were computed as described previously (Padlan,
`1990). Exposures were computed in the context of
`isolated Fvs.
`Residues in the interface between VL and VH in
`
`the KOL and 1539 Fabs were designated as being in
`contact if they have atoms that come within half an
`Angstrom of the sum of their van der Waals’ radii.
`
`(b) Sequence data
`
`A survey was made of the residues occurring at each
`position in the framework regions of human light and
`heavy chain variable domains. The sequences were
`
`obtained from the compilation of Kabat et a1. (1987).
`Only those sequences that are completely determined
`in the framework regions, 1—23, 35—49, and 57~88 in
`the light chains, and in the framework regions, 1—30,
`36—49, and 66—94, in the heavy chains (numbering of
`Kabat et al., 1987), were included in the survey. For
`the fourth framework region, 98—107 in the light chain
`and 103—1 13 in the heavy chain, only the residues that
`could be derived from the known J-minigene segments
`(Kabat et al., 1987) were considered. Here, the num-
`bering scheme of Kabat et al. (1987) is used through-
`out and pyrollidone carboxylic acids are counted as
`glutamines.
`The human kappa-chain sequences included in the
`survey were: ROY, AU, REI, HAU, HKlOl’CL,
`SCW, WEA, HK137’CL, HK134’CL, DAUDI’CL,
`WALKER’CL, GAL(1), LAY, WES, Vb’CL,
`HKIOZ’CL, EU, DEN, AMYLOID BAN, MEV,
`Vd’CL, Va’CL, KUE, Ve’CL, V13’CL, V18A’CL,
`Vl9A’CL, Vl9B’CL, and V18C’CL in V-kappa
`subgroup I; NIM, CUM, GM603’CL, FR, and
`RP Ml-6410’CL in V-kappa subgroup II; TI, WOL,
`SIE, NG9’CL, NEU, GOT, PAY, SON, GAR’, PIE,
`FLO, GLO, CUR, IARC/BL41’CL, POM, REE, and
`K-EVlS’CL in V-kappa subgroup III; and VJI’CL,
`VKAPPAIV GERMLINE’CL, PB17IV’CL,
`and
`LEN in V-kappa subgroup IV.
`The human lambda-chain sequences included in the
`survey were: NEWM, HA, NIG-64, NEW, BLZ’CL,
`WAH, NIG-77, VOR, RHE, LOC, OKA, COX, and
`NIG-51 in V-lambda subgroup I; NIG-84, MES,
`WH, NEI, WEIR, TOG, TRO, BOH, NIG-58, VIL,
`WIN, and 4A’CL in V-lambda subgroup II; HIL,
`LAP, GAR, and MOT in V-lambda subgroup III; SH
`in V—lambda subgroup IV (included for completeness
`although missing the residue at position 1); B0 and
`MCG in V-lambda subgroup V; AMYLOID-AR,
`SUT, THO, LBV’CL, and NIG-48 in V—lambda
`subgroup VI.
`The human heavy—chain sequences included in
`the survey were: EU, SIE, HG3’CL, WOL, ND’CL,
`and MOT in VH subgroup I; COR, DAW, OU,
`MCE’, CE—l’CL, HE, SUP-Tl VH-JA’CL, WAH,
`and HIGl’CL in VH subgroup II; TUR, LAMBDA-
`VH26/CL, POM, WAS, Hll’CL, TEI, BRO’IGM,
`LAY, GRA’, ZAP, JON, DOB, NIE, 333’CL,
`lHl’CL, lBll’CL, 126’CL, 112’CL,
`llS’CL, TRO,
`and KOL in VH subgroup III.
`
`(6) Comparison of representative mouse and human
`sequences
`
`The degree to which mouse variable domains
`would have to be mutated in order to “humanize”
`
`them, was determined by comparing the framework
`regions of representative sequences from the various
`mouse subgroups with the known human sequences
`(Kabat et al., 1987). The human sequence that should
`serve best for comparison purposes is that which is the
`most similar to the particular mouse sequence under
`consideration. However,
`in view of the possibility
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`Reduction of the immunogenicity of antibody variable domains
`
`491
`
`that individual sequences may have errors, the most
`frequently occurring residue at each position in the
`various human variable domain subgroups was also
`used in the comparisons. In many instances, more
`than one residue is found at high frequency at a given
`position; in those cases, any amino acid that occurs
`at least one-fourth as frequently as the most com—
`monly occurring residue was considered a possible
`alternative in the comparisons. The human subgroup
`to which each mouse sequence most closely corre-
`sponds was determined in this manner; the human
`sequence to which the mouse sequence is most similar
`was also determined.
`
`The sequences that were chosen as representative
`of the various mouse variable domain subgroups were:
`8107 (Subgroup I), TEPC(CAL20)105 (Subgroup II),
`TEPCl ll (Subgroup III), SlO7B’CL (Subgroup IV),
`R167 CRI+ (Subgroup V), and TEPC191 (Sub-
`group VI) of the kappa chains; MOPC104E of the
`lambda chains; and 36-60 CRI- (Subgroup I(A)),
`MCIOl/CL (Subgroup I(B)), MOPC104E (Subgroup
`II(A)), Bl-8’CL (Subgroup II(B)), G5 BB 2.2(AB1)
`Subgroup
`II(C)), TEPC15
`(Subgroup
`III(A)),
`XRPC44 (Subgroup III(B)), HPC76’CL (Subgroup
`III(C)), and 93G7 CRI +’CL (Subgroup V(A)) of the
`heavy chains (Kabat et al., 1987). These sequences are
`the first listed of the sequences in each subgroup in
`Kabat et al. (1987) that are completely determined in
`all four framework regions. None of the sequences in
`the VH subgroups V(A) and V(B) in the compilation
`of Kabat et a1.
`(1987) satisfies this criterion.
`In
`addition, MOPC21’CL was chosen to represent the
`miscellaneous mouse heavy chains which have not
`been assigned to any particular subgroup.
`
`RESULTS
`
`The solvent accessibility of the framework residues
`in the variable domains of KOL and .1539 and the
`
`amino acids most frequently occurring at each position
`in the various subgroups are given in Table 1 for heavy
`chains, in Table 2 for lambda chains, and in Table 3
`for kappa light chains. The residues that contact the
`opposite domain in the VL—VH interface are indi—
`cated in these tables. The results of the comparison
`of the mouse and human sequences are presented in
`Table 4.
`
`DISCUSSION
`
`A close examination of the fractional accessibility
`values presented in Table 1
`reveals a very close
`similarity in the exposure patterns of the VH domains
`of KOL and J539. Only at positions 88 and 104 are
`the two patterns drastically different and at these
`positions one or both proteins have glycine. According
`to the convention used here (see footnote to Table l),
`glycine is designated as being completely exposed if
`its alpha-carbon is accessible to the solvent probe,
`otherwise it is designated as being completely buried;
`
`thus, the slightest difference in structure could result
`in different exposure designations for homologous
`glycine residues. The exposure patterns of their VL
`domains (Tables 2 and 3) likewise are very similar
`with large differences only at positions 2, 13, 66, 99,
`and 101. The conformation of the amino-terminal
`
`segments of the KOL and J 539 light chains are
`slightly different, resulting in the difference observed
`at position 2; at the other positions, one or both
`proteins again have glycine.
`The very close similarity of the exposure patterns
`for the variable domains of KOL and J539 points to
`the close correspondence of the tertiary structures
`and of the dispositions of individual residues in these
`homologous domains. This is particularly remarkable
`because (a) these antibodies are from different species,
`(b) their light chains are of different types (.1539 has
`a kappa light chain, while KOL has a lambda light
`chain), (c) half of their CDRs, specifically the first
`and third CDRs in VL and the third in VH, have very
`different lengths and backbone conformations, and
`(d) KOL and J 539 have only 44 identical residues out
`of the 79 corresponding positions in the VL frame-
`work and 60 out of 87 in VH. An even closer similarity
`in overall structure and in the exposure patterns might
`be expected for two molecules that are more similar in
`sequence than this pair.
`By and large, the determination of which human
`variable domain subgroup an allogeneic domain most
`closely corresponds to is straightforward. However,
`in some of the human subgroups presented in Tables 1
`to 3, many positions exhibit several possible alterna-
`tives. Of the 76 positions in the first three framework
`regions in VH, 18 in Subgroup I presented more than
`one possible alternative for the amino acid compari-
`sons, 30 in Subgroup II, and 21 in Subgroup III. Of
`the 70 positions in the first three framework regions
`in the kappa light-chain variable domains, three in
`Subgroup I presented more than one possible alterna-
`tive, two in Subgroup 11, none in Subgroup III, and
`one in Subgroup IV; of the corresponding 69 positions
`in the lambda chains, 11 in Subgroup I presented more
`than one possible alternative, eight in Subgroup II,
`28 in Subgroup III, none in Subgroup IV since there
`is only one available sequence, eight in Subgroup V,
`and five in Subgroup VI. The existence of several
`possible alternatives in some of these subgroups,
`especially in the heavy chains, emphasizes the need for
`further subdivision; finer subgrouping would allow
`greater precision in the sequence correlations and
`greater ease in the identification of the residues which
`differ between human and non-human antibodies.
`
`A procedure is proposed here for reducing the
`antigenicity of an allogeneic variable domain while
`preserving its ligand-binding properties. It is based on
`the replacement of the residues which differ from
`those of the host with the corresponding residues in
`the most similar host sequence. Only those residues
`which are at least partly exposed in the corresponding
`domains of KOL or .1539 (those with pB, mE, or Ex
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`492
`
`E. A. PADLAN
`
`Table l. Solvent exposure of sidechains of framework residues in KOL and .1539 Fvs and the residues which occur most frequently at these
`positions in the various human VH subgroups
`
`Fractional accessibility
`KOL
`J539
`Residues in subgroup
`
`Position
`Residue
`Exposure
`Residue
`Exposure
`1
`II
`III
`
`1
`2
`3
`4
`5
`6
`7
`8
`9
`10
`11
`12
`13
`14
`15
`16
`17
`18
`19
`20
`21
`22
`23
`24
`25
`26
`27
`28
`29
`30
`36
`37
`38
`39
`40
`41
`42
`43
`44
`45
`46
`47
`48
`49
`66
`67
`68
`69
`70
`71
`72
`73
`74
`75
`76
`77
`78
`79
`80
`81
`82
`8221
`82b
`82c
`83
`84
`85
`86
`87
`88
`89
`90
`91
`92
`93
`94
`
`E
`V
`Q
`L
`V
`Q
`S
`G
`G
`G
`V
`V
`Q
`P
`G
`R
`S
`L
`R
`L
`S
`C
`S
`S
`S
`G
`F
`I
`F
`S
`W
`V
`R
`Q
`A
`P
`G
`K
`G
`L
`E
`W
`V
`A
`R
`F
`T
`I
`S
`R
`N
`D
`S
`K
`N
`T
`L
`F
`L
`Q
`M
`D
`S
`L
`R
`P
`E
`D
`T
`G
`V
`Y
`F
`C
`A
`R
`
`1.00 Ex
`0.23 mB
`0.82 Ex
`0.00 Bu
`0.87 Ex
`0.00 Bu
`0.94 Ex
`1.00 Ex
`0.00 Bu
`1.00Ex
`0.90 Ex
`0.25 1113
`0.71 mE
`0.59 pB
`1.00 Ex
`0.73 mE
`0.66 mE
`0.28 ml!
`0.66 mE
`0.00 Bu
`0.71 mE
`0.00 Bu
`1.00 Ex
`0.00 Bu
`0.87 Ex
`100 Ex
`0.IOBu
`0.85 Ex
`0.00 Bu
`0.74 mE
`0.00 Bu
`0.00 Bu
`0.10 Bu
`0.15 Bu
`0.95 Ex
`0.90 Ex
`1.00 Ex
`0.86 Ex
`1.00 Ex
`0.00 Bu
`0.75 mE
`0.10 Bu
`0.00 Bu
`0.00 Bu
`0.36 mB
`0.00 Bu
`0.87 Ex
`0.00 Bu
`0.78 mE
`0.11 Bu
`0.61 mE
`044 p8
`0.85 Ex
`0.88 Ex
`0.69 mE
`0.41pB
`0.00 Bu
`045 p3
`0.00 Bu
`0.53 pH
`0.00 Bu
`0.73mE
`0.98 Ex
`0.00 Bu
`0.73 mE
`075 Me
`0.82 Ex
`0.00 Bu
`0.54 pB
`1.00 Ex
`0.58 pH
`0.00 Bu
`0.00 Bu
`0.00 Bu
`0.00 Bu
`0.]7Bu
`
`E
`V
`K
`L
`L
`E
`S
`G
`G
`G
`L
`V
`Q
`P
`G
`G
`S
`L
`K
`L
`S
`C
`A
`A
`S
`G
`F
`D
`F
`S
`W
`V
`R
`Q
`A
`P
`G
`K
`G
`L
`E
`W
`1
`G
`K
`F
`I
`1
`S
`R
`D
`N
`A
`K
`N
`S
`L
`Y
`L
`Q
`M
`S
`K
`V
`R
`S
`E
`D
`T
`A
`L
`Y
`Y
`C
`A
`R
`
`1.00 Ex
`0.37 mB
`0.82 Ex
`0.10 Bu
`1.00 Ex
`0.09 Bu
`0.94 Ex
`1.00 Ex
`0.00 Bu
`1.00Ex
`0.81 Ex
`025 m3
`0.87 Ex
`0.64 mE
`1.00 Ex
`1.00 Ex
`0.75 mE
`0.26 mB
`0.75 mE
`0.00 Bu
`0.82 Ex
`0.00 Bu
`100 EX
`0.00 Bu
`1.00 Ex
`1.00 Ex
`0.10Bu
`0.72 mE
`0.00 Bu
`0.83 Ex
`0.00 Bu
`0.00 Bu
`0.31 mB
`0.28 mB
`0.75 mE
`0.73 mE
`1.00 Ex
`0.86 Ex
`1.00 Ex
`0.00 Bu
`0.73 mE
`0.04 Bu
`0.00 Bu
`0.00 Bu
`0.51 pH
`0.00 Bu
`0.88 Ex
`0.00 Bu
`0.79 mE
`0.00Bu
`0.55 pB
`0.43 pH
`0.97 Ex
`0.77 mE
`0.68 mE
`0.33 mB
`0.00 Bu
`0.35 mB
`0.00 Bu
`0.69mE
`0.00 Bu
`0.58pB
`0.96 Ex
`0.00 Bu
`0.83 Ex
`0.90 Ex
`0.90 Ex
`0.11 Bu
`0.47 pB
`0.00 Bu
`0.63 mE
`0.00 Bu
`0.08 Bu
`0.00 Bu
`0.00 Bu
`0.158u
`
`Q
`V
`Q
`L
`V
`Q
`S
`G
`A
`E
`V
`K
`K
`P
`G
`SA
`S
`V
`R K
`V
`S
`C
`K
`A T V
`S
`G
`GYD
`T
`F
`S N V1
`W
`V
`R
`Q
`A
`P
`G
`Q R K H
`G
`L
`E
`W
`M V
`G
`R
`V
`T
`V M l
`TS
`RLA
`D K
`P E T A S
`S
`T F
`N S T
`TQ
`A V
`Y
`M
`E
`L
`SVRT
`S
`L
`R F1
`S
`E
`D
`T
`A
`V
`Y
`Y
`C
`A
`R
`
`Q
`V
`T Q
`L
`RQKT
`E
`S
`G
`P
`AGT
`L
`V
`K
`P
`TS
`EQ
`T
`L
`T S
`L
`T
`C
`T
`F V
`S
`G
`FLG
`S
`L I
`S
`W
`1
`R
`Q
`P
`P
`G
`K R
`A G
`L
`E
`W
`L I
`A G
`R
`L V
`T
`I V
`ST
`KV
`D
`T
`S
`K R
`N
`Q
`V F
`V S
`L
`TKSIN
`M L
`TSNIR
`N S
`V M
`D T
`P A
`V A
`D
`T
`A
`T V
`Y
`Y
`C
`A
`RH
`
`E
`V M
`Q
`L
`VL
`E
`S
`G
`G
`GA
`L F
`V
`Q
`P
`G
`G
`S
`L
`R K
`L
`S
`C
`A
`A
`S
`G
`F
`T N
`F
`S
`W
`V
`R
`Q
`A
`P S
`G
`K
`G S
`L
`E
`W
`V
`G S A
`R
`F
`T
`I
`S
`R
`D N
`D N
`S
`K
`N
`T
`L A
`Y F
`L
`Q
`M
`ND
`S
`L
`R E
`P A
`E D
`D
`T
`A
`V L
`Y
`Y
`C
`AT
`RP
`
`4 of 1 0
`
`Bl Exhibit 1118
`
`4 of 10
`
`BI Exhibit 1118
`
`

`

`Reduction of the immunogenicity of antibody variable domains
`
`493
`
`Table l~con£inaed
`
`Fractional accessibility
`
`
` KOL 1539 Residues in subgroup
`Position
`Residue
`Exposure
`Residue
`Exposure
`I
`II
`III
`JI-Il
`IH2
`JH3
`1H4
`JHS
`JI-I6
`W
`W
`W
`W
`W
`W
`0.07 Bu
`W
`0.09 Bu
`W
`103
`G
`G
`G
`G
`G
`G
`100 Ex
`G
`0.00 Bu
`C
`104
`Q
`R
`Q
`Q
`Q
`Q
`099 EX
`Q
`0.93 Ex
`Q
`105
`G
`G
`G
`G
`G
`G
`000 Bu
`G
`0.00 Bu
`G
`106
`T
`T
`T
`T
`T
`T
`0.26 ml;
`T
`0.22 ml?
`T
`l0?
`L
`L
`M
`L
`L
`T
`0.67 ml?
`L
`099 EX
`P
`108
`V
`V
`V
`V
`V
`V
`0.00 Bu
`V
`0.00 Bu
`V
`109
`T
`T
`T
`T
`T
`T
`0.69 mE
`T
`0.?6 mE
`T
`HO
`V
`V
`V
`V
`V
`V
`0.00 Bu
`V
`0.00 Bu
`V
`111
`S
`S
`S
`S
`S
`S
`0.74 mE
`5
`0.98 Ex
`S
`112
`
`
`S113 S 0.94 Ex A 0.84 Ex S S S S S
`
`
`
`
`
`
`
`
`
`The one-letter amino acid code is used here. The fractional solvent accessibility values for the individual residues were computed as described
`by Padlan (1990); residues whose sidechains have fractional accessibility values between 0.00 and 0.20 are designated as being completely
`buried (Bu), between 0.20 and 0.40 as mostly buried (1113), between 0.40 and 0.60 as partly buried/partly exposed (p13), between 0.60
`and 0.80 as mostly exposed (mE), and at least 0.80 as completely exposed (Ex). In the special case of glycine, the residue is considered
`completely exposed if its alpha-carbon atom is accessible to solvent, otherwise it is considered completely buried. Residues that are
`involved in the VL—VH contact are shown in bold letters.
`
`Table 2. Solvent exposure of sidechains of framework residues in KOL VL and the residues which occur most frequently at these positions
`“WWW
`in the various human V-lambda subgroups
`Residues in subgroup
` Position Residue Exposure I II III IV V VI
`
`
`
`
`
`
`
`
`1
`Q
`1.00 Ex
`Q
`Q
`S F
`—-
`Q
`N D
`2
`S
`1.00 Ex
`S
`S
`Y
`S
`S
`F
`3
`V
`0.77 mE
`V
`A
`E
`E
`A
`M
`4
`L
`0.00 Bu
`L
`L
`L
`L
`L
`L
`5
`T
`0.92 Ex
`T
`T
`T K
`T
`T
`T
`6
`Q
`0‘30 BU
`Q
`Q
`Q
`Q
`Q
`Q
`7
`P
`0.62 mE
`P
`P
`P
`D
`P
`P
`8
`P
`1.00 Ex
`P
`A R P
`P
`P
`P
`H
`9
`S
`1.00 Ex
`S
`S
`S
`A
`S
`S
`10
`_
`-~— -
`—
`—
`—
`—
`—
`-—
`11
`A
`0.34 ml!
`A V
`V
`V
`V
`A
`V
`12
`S
`0.71 mE
`S
`S
`S
`S
`S
`S
`13
`G
`1.00 Ex
`G A
`G
`V L
`V
`G
`E
`14
`T
`0.73 mE
`T A
`S
`S A
`A
`S
`S
`15
`P
`0.75 mE
`P
`P
`P A
`L
`P L
`P
`16
`G
`1.00 Ex
`G
`G
`G
`G
`G
`G
`17
`Q
`0.69 m5
`Q
`Q
`Q
`Q
`Q
`K
`18
`R
`0.79 mE
`R
`S
`T
`T
`S
`T
`19
`V
`021 1118
`V
`IV
`A
`V
`V
`V
`20
`T
`0.62 mE
`T
`T
`R M
`R
`T
`T
`21
`I
`0.00 Bu
`I
`I
`I
`I
`I
`I F M
`22
`S
`0.92 Ex
`S
`S
`T
`T
`S
`S
`23
`C
`0.00 Bu
`C
`C
`C
`C
`C
`C
`35
`W
`0.00 Bu
`W
`W
`W
`W
`W
`W
`36
`Y
`0.00 Bu
`Y
`Y F
`Y
`Y
`Y
`Y
`37
`Q
`0.46 p3
`Q
`Q
`Q
`Q
`Q
`Q
`38
`‘
`0.00 Bu
`Q H
`Q
`Q E
`Q
`Q
`Q
`39
`..
`0.75 mE
`L V
`H
`K R
`K
`H
`R
`40
`P
`0.91 Ex
`P
`P
`P S
`P
`P A
`P
`41
`G
`1.00 Ex
`G
`G
`G
`G
`G
`G
`42
`M
`0.74 mE
`T
`K
`Q R
`Q
`R K
`S R G
`43
`A
`0.62 ml?
`A
`A
`A
`A
`A
`A
`44
`P
`0.00 Bu
`P
`P
`P
`P
`P
`P
`45
`K
`0.95 Ex
`K
`K
`V
`L
`K
`T
`46
`L
`0.23 mB
`L
`L
`M L P
`L
`L V
`T
`47
`L
`0.15 Bu
`L
`M l L
`V
`V
`V I
`V
`48
`I
`0.00 Bu
`I
`I
`IV
`I
`I
`I
`49
`Y
`0.39 ml?!
`Y
`Y F
`Y
`Y
`F Y
`Y
`57
`G
`100 Ex
`G
`G
`G E
`G
`G
`G
`58
`V
`0.14 Bu
`VI
`V I
`IV
`I
`V
`V
`59
`P
`0.70 mE
`P
`S P
`P
`P
`P
`P
`60
`D
`0.95 Ex
`D
`D N L
`E Q A
`D
`D
`D
`61
`R
`0.31 m3
`R
`R
`R
`R
`R
`R
`62
`F
`0.12 Bu
`F
`F
`F
`F
`F
`F
`63
`S
`0.85 Ex
`S
`S
`S
`S
`S
`S
`64
`G
`0.00 Bu
`G A
`G
`G S
`G
`G
`G
`65
`S
`1.00 Ex
`S
`S
`S Y
`S
`S
`S
`66
`K
`0.41 138
`K
`K
`T S N
`S
`K
`I F‘
`67
`S
`1.00 Ex
`S
`S
`S
`S
`S
`S
`68
`G
`1.00 Ex
`G
`G
`G
`G
`D G
`S
`69
`A
`0.71 mE
`T
`N
`T N
`H
`N
`N
`
`(continued averted)” )
`
`5of10
`
`BI Exhibit1118
`
`5 of 10
`
`BI Exhibit 1118
`
`

`

`494
`
`E. A. PADLAN
`
`Table 2—c0ntinued
`
`Residues in subgroup
`
`III
`IV
`Position
`Residue
`Exposure
`I
`II
`V
`VI
`70
`S
`1.00 Ex
`S
`T
`T K S
`T
`T
`S
`71
`A
`0.00 Bu
`A
`A
`A V
`A
`A
`A
`72
`S
`1.00 Ex
`S T
`S
`TI
`S
`S
`S
`73
`L
`0.00 Bu
`L
`L
`L
`L
`L
`L
`74
`A
`0.74 mE
`A
`T
`T
`T
`T
`T
`75
`I
`0.00 Bu
`I
`I
`I
`I
`V
`I
`76
`G
`1.00 Ex
`S T
`S
`S N
`T
`S
`S
`77
`G
`1.00 Ex
`G
`G
`G R
`G
`G
`G
`78
`L
`0.00 Bu
`L
`L
`V A
`A
`L
`L
`79
`Q
`0.76mE
`QR
`Q
`QE
`Q
`RQ
`KQT
`80
`S
`100 EX
`S T
`A
`A V
`A
`A
`T
`81
`E
`0.78 mE
`E G
`E
`E G
`E
`E
`E
`82
`D
`0.09 Bu
`D
`D
`D
`D
`D
`D
`83
`E
`0.64 mE
`E
`E
`E
`E
`E
`E
`84
`T
`0.34 mB
`A
`A
`A
`A
`A
`A
`85
`D
`0.30 mB
`D
`D
`D
`D
`D
`D
`86
`Y
`0.00 Bu
`Y
`Y
`Y
`Y
`Y
`Y
`87
`Y
`0.16 Bu
`Y
`Y
`Y F
`Y
`Y
`Y
`88
`C
`0.00 Bu
`C
`C
`C
`C
`C
`C
`JL-l
`JL—2
`JL-3
`JL—4
`JL-5
`F
`F
`F
`F
`F
`0.04 Bu
`F
`98
`G
`G
`G
`G
`G
`0.00 Bu
`G
`99
`T
`G
`G
`S
`S
`0.59 pH
`T
`100
`G
`G
`G
`G
`G
`1.00 Ex
`G
`101
`T
`T
`T
`T
`T
`0.00 Bu
`T
`102
`K
`K
`K
`Q
`Q
`0.82 Ex
`K
`103
`V
`L
`L
`L
`L
`0.00 Bu
`V
`104
`T
`T
`T
`T
`T
`086 EX
`T
`105
`V
`V
`V
`V
`V
`0.19 Bu
`V
`106
`106a
`L
`0.70 mE
`L
`L
`L
`L
`L
`
`107
`G
`1.00 Ex
`G
`G
`G
`S
`G
`*Additional residues after position 66:
`66a D
`6613 S R D.
`(see footnote to Table 1).
`
`Table 3. Solvent exposure of sidechains of framework residues in J539 VL and the residues which occur most frequently at these positions
`in the various human V-kappa subgroups
`Residues in subgroup
`Residues in subgroup
`
`Position Residue
`Exposure
`I
`II
`III
`IV
`Position Residue
`Exposure
`I
`II
`III
`IV
`1
`E
`0.99 Ex
`D
`D
`E
`D
`48
`I
`0.00 Bu
`I
`I
`I
`I
`2
`I
`0.16 Bu
`I
`I
`I
`I
`49
`Y
`0.42 pH
`Y
`Y
`Y
`Y
`3
`V
`0.87 Ex
`Q
`V
`V
`V
`57
`G
`1.00 Ex
`G
`G
`G
`G
`4
`L
`0.00 Bu
`M
`M
`L
`M
`58
`V
`0.13 Bu
`V
`V
`I
`V
`5
`T
`0.80 mE
`T
`T
`T
`T
`59
`P
`0.61 mE
`P
`P
`P
`P
`6
`Q
`0.00 Bu
`Q
`Q
`Q
`Q
`60
`A
`1.00 Ex
`5
`D
`D
`D
`7
`S
`0.89 Ex
`S
`S
`S
`S
`61
`R
`0.36 ml!
`R
`R
`R
`R
`8
`P
`0.67 mE
`P
`P
`P
`P
`62
`F
`0.00 Bu
`F
`F
`F
`F
`9
`A
`1.00 Ex
`5
`L
`G
`D N
`63
`S
`0.94 Ex
`S
`S
`S
`S
`10
`I
`094 EX
`S
`S
`T
`S
`64
`G
`0.00 Bu
`G
`G
`G
`G
`11
`T
`0.30 mB
`L
`L
`L
`L
`65
`S
`1.00 Ex
`S
`S
`S
`S
`12
`A
`0.59 pH
`S
`P
`S
`A
`66
`G
`1.00 Ex
`G
`G
`G
`G
`13
`A
`0.00 Bu
`A
`V
`L
`V
`67
`S
`1.00 Ex
`S
`S
`S
`S
`14
`S
`0.78 mE
`S
`T
`S
`S
`68
`G
`1.00 Ex
`G
`G
`G
`G
`15
`L
`0.79 mE
`V
`P
`P
`L
`69
`T
`0.75 mE
`T
`T
`T
`T
`16
`G
`1.00 Ex
`G
`G
`G
`G
`70
`S
`098 EX
`D EQ D
`D
`D
`17
`Q
`0.64 mE
`D
`E
`E
`E
`71
`Y
`0.09 Bu
`F
`F
`F
`F
`18
`K
`0.74 mE
`R
`P
`R
`R
`72
`S
`0.70 mE
`T
`T
`T
`T
`19
`V
`022 m8
`V
`A
`A
`A
`73
`L
`0.00 Bu
`L
`L
`L
`L
`20
`T
`0.65 mE
`T
`S
`T
`T
`74
`T
`0.43 pH
`T
`K
`T
`T
`2|
`I
`0.00 Bu
`I
`I
`L
`I
`75
`I
`0.00 Bu
`I
`I
`I
`I
`22
`T
`0.69 mE
`T
`S
`S
`N
`76
`N
`0.83 Ex
`S
`S
`S
`S
`23
`C
`0.00 Bu
`C
`C
`C
`C
`77
`T
`0.83 Ex
`S
`R
`R
`S
`35
`W
`0.00 Bu
`W
`W
`W
`W
`78
`M
`0.00 Bu
`L
`V
`L
`L
`36
`Y
`0.00 Bu
`Y
`Y
`Y
`Y
`79
`E
`0.63 mE
`Q
`E Q
`E
`Q
`37
`Q
`0.14 Bu
`Q
`L
`Q
`Q
`80
`A
`0.96 Ex
`P
`A
`P
`A
`38
`Q
`024 m8
`Q
`Q
`Q
`Q
`81
`E
`0.91 Ex
`E D
`E
`E
`E
`39
`K
`0.69 mE
`K
`K
`K
`K
`82
`D
`0.13 Bu
`D
`D
`D
`D
`40
`S
`1.00 Ex
`P
`P
`P
`P
`83
`A
`0.55 pH
`F I
`V
`F
`V
`41
`G
`1.00 Ex
`G
`G
`G
`G
`84
`A
`0.00 Bu
`A
`G
`A
`A
`42
`T
`0.90 Ex
`K
`Q
`Q
`Q
`85
`I
`0.58 pB
`T
`V
`V
`v
`43
`S
`0.30 mB
`A
`S
`A
`P
`86
`Y
`0.00 Bu
`Y
`Y
`Y
`Y
`44
`P
`0.00 Bu
`P
`P
`P
`P
`87
`Y
`0.11 Bu
`Y
`Y
`Y
`Y
`45
`K
`090 EX
`K
`Q E R R
`K
`88
`C
`0.00 Bu
`C
`C
`C
`C
`46
`P
`0.43 pH
`L
`L
`L
`L
`47
`W
`0.16 Bu
`L
`L
`L
`L
`
`(continued next page)
`
`60f10
`
`BI Exhibit1118
`
`6 of 10
`
`BI Exhibit 1118
`
`

`

`Reduction of the immunogenicity of antibody variable domains
`
`495
`
`Table 3—continued
`
`Residues in subgroup
`
`I
`II
`III
`Position Residue
`Exposure
`IV
`JK-l
`JK-2 JK-3 JK-4 JK-5
`F
`F
`F
`F
`F
`0.00 Bu
`F
`98
`G
`G
`G
`G
`G
`1.00 Ex
`G
`99
`Q
`Q
`P
`G
`Q
`1.00 Ex
`A
`100
`G
`G
`G
`G
`G
`0.00 Bu
`G
`101
`T
`T
`T
`T
`T
`0.00 Bu
`T
`102
`K
`K
`K
`K
`R
`0.79 mE
`K
`103
`V
`L
`V
`V
`L
`0.00 Bu
`L
`104
`E
`0.89 Ex
`E
`E
`D
`E
`E
`105
`
`L
`0.44 pH
`1
`I
`I
`I
`I
`106
`106a
`
`K107 K 0.77 mE K K K K
`
`
`(see footnote to Table 1).
`
`
`
`
`
`
`
`
`
`designations in Tables 1—3) are replaced. One would
`retain (a) the CDRs, (b) the residues in the immediate
`neighborhood of the CDRs (the last
`framework
`residue before and the first after each CDR might be
`enough),
`(c)
`the residues corresponding to those
`which in KOL and J539 are completely or mostly
`buried (mB and Bu designations in Tables 1-3), and
`(d) the residues corresponding to those which in KOL
`or J 539 are involved in the interdomain contact.
`
`the number of
`On the basis of this proposal,
`residues in a mouse framework that would need to be
`replaced to “humanize” it can be determined for each
`representative mouse variable domain. This number
`is found to range from 7 to 16, when the mouse
`sequences are correlated with the most frequently
`occurring residues in the human variable domain
`subgroups, and from 6 to 15 when they are compared
`with the human sequences to which they are most
`similar (Table 4). These numbers are not large and,
`since in many instances the differences are bunched
`
`(data not shown, but see examples below), the amino
`acid replacements by site-directed mutagenesis would
`not be too difficult.
`
`In some of the cases presented in Table 4, many
`buried and contacting residues are found to be differ-
`ent in the mouse and human domains. In those cases,
`unless the interior and contacting residues are also
`replaced, simple grafting of the CDRs to a human
`framework could result in alterations in ligand-binding
`properties.
`One position at which human and mouse sequences
`frequently differ is the N-terminus in both chains.
`The N-termini are contiguous with the CDR surface
`and are in position to be involved in ligand binding.
`It might therefore be wise to keep the N-terminal
`residues of the original antibodies in the “humanized“
`ones.
`
`The replacement of some amino acid types could
`have a significant effect on the tertiary structure of
`the domains. For example, the replacement of proline
`by another amino acid and vice versa, could cause
`a restructuring of the local region;
`this could be
`particularly serious if the proline is
`in the cis
`configuration. A change in the local structure could
`also occur following the replacement of a glycine by
`another amino acid type, if the backbone dihedral
`angles of the glycine are energetically unfavorable for
`other types of residues.
`A replacement that could have a significant effect
`on ligand binding, even if it
`is distant from the
`combining site region, is one which involves a change
`in electrical charge. If electrostatic interactions have
`been determined to be important in the binding of the
`antigen, replacements should be chosen judiciously
`
`Table 4. Comparison of the frameworks of representative mouse and human variable domains
`Difierences’r
`vs
`
`RPM1-6410’CL
`
`V19A‘CL
`
`VLIII, JL2
`
`4A’CL
`
`20
`10
`15
`26
`15
`25
`
`29
`
`5
`3
`4
`9
`5
`8
`
`2"
`1"
`
`2 + 1““
`3'
`l + 1*
`
`10
`
`3 + 4‘
`
`15
`7
`11
`15
`10
`l6
`
`16
`
`8
`
`21
`
`231
`
`2
`
`4
`
`9
`
`1"
`
`2 + 1"
`
`6
`
`15
`
`3 + 5"
`
`10
`
`(most similar sequence)
`(most similar subgroup)
`Need
`Need
`Mouse
`Most similar human:
`Con-
`to
`Con-
`to
`
`sequence
`Subgroup
`Sequence
`Total Buried
`tacting
`replace
`Total Buried
`tacting
`replace
`VL (kappa):
`VKIV, JKZ
`$107
`TEPC(CAL20)105 VKII, JK4
`TEPClll
`VKIV,JK4
`SlO7B’CL
`VKIV, JKZ
`R16.7 CRI+
`VKI,JK1
`TEPC191
`VKIV, JK2
`VL (lambda):
`MOPC104E
`VII:
`15
`10
`271
`11
`10
`231:
`SUP-TI-VH-JA’CL
`VHII, JI-I4
`36—60 CRI-
`14
`13
`281
`11
`6
`181
`SUP-Tl-VH—JA’CL
`VHII,JH4
`MC101’CL
`12
`10
`251
`11
`9
`231
`HGB’CL
`VH1, JH6
`MOPC104E
`11
`9
`21:
`10
`9
`20:
`HG3’CL
`VH1, JH6
`Bl-8’CL
`11
`13
`25:
`11
`9
`211
`HGS’CL
`VHI, JH4
`G5 BB 2t2(ABl)
`8
`6
`16
`8
`4
`14
`LAMBDA«VH26’CL
`VHIII, JH6
`TEPC15
`10
`5
`15
`8
`3
`11
`LAMBDA-VH26’CL
`VHIII, JH4
`XRPC44
`10
`7
`17
`8
`5
`13
`LAMBDA-VI—I26’CL
`VHIII, JH6
`I—IPC76’CL
`14
`11
`261
`12
`10
`231
`I-IG3’CL
`VHI, JH4
`93G7 CR1 + ’CL
`
`MOPC21’CL 9 VHIII, JI-I6 H1 1’CL 8 1 7 12 3
`
`
`
`
`
`
`
`
`1 + 2‘
`1‘
`2
`
`2
`
`l + 2‘
`1"
`2
`
`2
`
`When no human sequence is given, it means that none is more similar to the mouse sequence than the consensus subgroup sequence.
`*Those contacting residues are buried.
`'l'The differences in the J-regions have been included.
`iThese include a residue that borders a CDR.
`
`7of10
`
`BI Exhibit 1118
`
`7 of 10
`
`BI Exhibit 1118
`
`

`

`496
`
`E. A. PADLAN
`
`
`
`Fig. 1. Stereodrawing of the alpha-carbon backbone trace of J539 Fv. The N - and C -terminal residues
`are labeled, as are every 20th residue in the sequence (numbering of Kabat et al., 1987). The VH domain
`is on the left and VL is on the right. The residues, which need to be replaced in the variable domains
`of the mouse antibody to “humanize” them, are indicated by the large open circles for $107 (a) and for
`MOPC21 (b).
`
`to preserve as much as possible the density and
`distribution of electrical charges on the molecule.
`The application of the procedure is demonstrated
`by the following examples. In these examples, only
`the use of the most frequently occurring residues in
`the various human variable domain subgroups and
`J-minigenes is explored.
`
`(a) A “worst-case” example
`
`Among the mouse kappa VLs listed in Table 4, for
`which the autologous VH had also been sequenced,
`SlO7 VL has the most residues that need to be replaced
`to “humanize” it. $107 VL is most similar in its
`
`framework to the members of the human subgroup
`VKIV and JK2, from which it difl'ers at positions 9,
`10, 14, 15, 16, 18, 19, 22, 38, 41, 43, 63, 78, 80, 83,
`84, 85, 100, and 106. Of these, positions 19, 38, 43,
`78, and 84 are occupied by residues that in J 539 are
`buried (Table 3); moreover, the residues at positions
`38 and 43 are involved in interdomain contact in
`1539. Thus the residues in $107 VL that need to be
`
`l7,
`l6,
`replaced are those at positions 9, 10, 14, 15,
`18, 22, 41, 63, 80, 83, 85, 100, and 106. $107 VH
`(Kabat et al., 1987) is most similar in its framework
`to the members of the human subgroup VHIII and
`JH6, from which it diflers at positions 3, 24, 40, 44,
`48, 68, 69, 73, 75, 76, 77, 82b, and 105. Of these,
`positions 24, 48, 69, and 77 are occupied by residues
`that in J539 or KOL are buried (Table 1); in addition,
`the residues at positions 44 and 105 are involved in
`interdomain contacts in 1539 or KOL. Thus the
`
`residues in S107 VH that need to be replaced are those
`at positions 3, 40, 68, 73, 75, 76, 82b, and 89. The 23
`residues that need to be replaced to “humanize” the
`variable domains of S107 are indicated in Fig. 1(a).
`
`(b) A “best-case” example
`
`Among the mouse VH included in Table 4, for
`which the autologous VL had also been sequenced,
`MOPC21 has the least number of residues that need
`to be replaced to “humanize” it. MOPC21 VH is
`most similar in its framework to the members of the
`
`8of10
`
`BIEmeH1118
`
`8 of 10
`
`BI Exhibit 1118
`
`

`

`Reduction of the immunogenicity of antibody variable domains
`
`497
`
`human subgroup VHIll and JH6 from which it
`differs at positions 1, 18, 42, 74, 823, 84, 89, and 108.
`Of these, the residue at position 18 is buried. Thus,
`only the residues at positions 1, 42, 74, 82a, 84,
`89, and 108 need to be replaced in MOPC21 VH.
`MOPCZI VL (Kabat et al., 1987) is most similar in
`its framework to the human subgroup VKIV and
`JK4, from which it differs at positions 1, 9, 11, 12, 13,
`15, 19, 21, 22, 41, 43, 63, 68, 78, 83, 85, 87, and 104.
`Of these, positions ll, 13, 19, 21, 43, 78, 87, and 104
`are occupied by residues that in J539 are buried
`(Table 3); moreover, the residues at positions 43 and
`87 are involved in the interdomain contact. Thus, the
`residues in MOPC21 VL that need to be replaced are
`those at positions 1, 9, 12, 15, 22, 41, 63, 68, 83,
`and 85. The 17 residues that need to be replaced to
`“humanize" the variable domains of MO