Proc. Natl. Acad. Sci. USA
`Vol. 89, pp. 4285-4289, May 1992
`Immunology
`
`Humanization of an anti-p18
`cancer therapy
`(antibody engineering/site-directed mutagenesis/c-erbB-2/neu)
`
`5HER2 antibody for human
`
`PAUL CARTER*, LEN PRESTA*, CORNELIA M. GorMANT, JOHN B. B. RIDGWAYt, DENNIS HENNER’,
`Wal LEE T. WonG?, ANN M. RowLaAnp}, CLairRE Kotts?, MONIQUE E. CARVER},
`AND H. MICHAEL SHEPARDS
`
`Departments of *Protein Engineering, tCell Genetics, *Medicinal and Analytical Chemistry, and §Cell Biology, Genentech Inc., 460 Point San Bruno
`Boulevard, South San Francisco, CA 94080
`
`Communicated by Hilary Koprowski, January 16, 1992 (received for review February 15, 1991)
`
`The murine monoclonal antibody mumAb4D5,
`ABSTRACT
`directed against human epidermal growth factor receptor 2
`(p185"=®2), specifically inhibits proliferation of human tumor
`cells overexpressing p185=?, However,
`the efficacy of
`mumAb4D5 in human cancer therapyis likely to be limited by a
`human anti-mouse antibody response and lack of effector func-
`tions. A ‘“‘humanized”’ antibody, humAb4D5-1, containing only
`the antigen binding loops from mumADb4D5 and human variable
`region framework residues plus IgG1 constant domains was
`constructed. Light- and heavy-chain variable regions were simul-
`taneously humanized in one step by ‘‘gene conversion mutagen-
`esis”’ using 311-mer and 361-mer preassembled oligonucleotides,
`respectively. The humAb4D5-1 variant does not block the pro-
`liferation of human breast carcinoma SK-BR-3 cells, which
`overexpress p185"£®2, despite tight antigen binding (Ka = 25
`nM). One of seven additional humanized variants designed by
`molecular modeling (humAb4D5-8) binds the p185"**? antigen
`250-fold and 3-fold more tightly than humAb4D5-1 and
`mumAb4D5,respectively. In addition, humAb4D5-8 has potency
`comparable to the murine antibody in blocking SK-BR-3 cell
`proliferation. Furthermore, humAb4D5-8 is much more efficient
`in supporting antibody-dependent cellular cytotoxicity against
`SK-BR-3 cells than mumAb4D5,but it does not efficiently kill
`WI-38 cells, which express p185"£"? at lower levels.
`
`The protooncogene HER2 encodesa protein tyrosine kinase
`(p185#ER2)
`that
`is homologous to the human epidermal
`growth factor receptor (1-3). Amplification and/or overex-
`pression of HER2is associated with multiple human malig-
`nancies and appearsto be integrally involved in progression
`of 25-30% of human breast and ovarian cancers (4, 5).
`Furthermore, the extent of amplification is inversely corre-
`lated with the observed medianpatient survival time (5). The
`murine monoclonal antibody mumAb4DS (6), directed
`against the extracellular domain (ECD)of p185#?, specif-
`ically inhibits the growth of tumorcell lines overexpressing
`p185#£®? in monolayer culture or in soft agar (7, 8).
`mumAb4DSalso has the potential of enhancing tumorcell
`sensitivity to tumornecrosis factor (7, 9). Thus, mumAb4D5
`has potential for clinical intervention in carcinomasinvolving
`the overexpression of p185#=®?,
`A majorlimitation in the clinical use of rodent mAbsis an
`anti-globulin response during therapy (10, 11). A partial
`solution to this problemis to construct chimeric antibodies by
`coupling the rodent antigen-binding variable (V) domains to
`human constant (C) domains (12-14). The isotype of the
`human C domains may be varied to tailor the chimeric
`antibody for participation in antibody-dependent cellular
`
`The publication costs of this article were defrayed in part by page charge
`payment.This article must therefore be hereby marked ‘‘advertisement’’
`in accordance with 18 U.S.C. §1734 solely to indicate this fact.
`
`cytotoxicity (ADCC) and complement-dependentcytotoxic-
`ity (CDC) (15). Such chimeric antibody moleculesarestill
`=~30% rodent in sequence and are capable ofeliciting a
`significant anti-globulin response.
`Winter and coworkers (16-18) pioneered the ‘‘humaniza-
`tion’’ of antibody V domains by transplanting the comple-
`mentarity determining regions (CDRs), which are the hyper-
`variable loops involved in antigen binding, from rodent
`antibodies into human V domains. Thevalidity of this ap-
`proach is supported bytheclinical efficacy of a humanized
`antibody specific for the CAMPATH-1 antigen with two
`non-Hodgkin lymphoma patients, one of whom had previ-
`ously developed an anti-globulin response to the parental rat
`antibody (17, 19). In some cases, transplanting hypervariable
`loops from rodent antibodies into human frameworks is
`sufficient to transfer high antigen binding affinity (16, 18),
`whereasin othercasesit has been necessary to also replace
`one(17) or several (20) framework region (FR) residues. For
`a given antibody, a small number of FR residuesare antici-
`pated to be important for antigen binding. First, there are a
`few FR residues that directly contact antigen in crystal
`structures of antibody-antigen complexes (21). Second, a
`number of FR residues have been proposed (22-24) as
`critically affecting the conformation of particular CDRs and
`thus their contribution to antigen binding.
`Here wereport the rapid and simultaneous humanization of
`heavy-chain (Vy) and light-chain (V,) V region genes of
`mumAb4DSbyusing a ‘‘gene conversion mutagenesis’’ strat-
`egy (43). Eight humanized variants (humAb4DS5) were con-
`structed to probe the importance of several FR residues
`identified by our molecular modeling or previously by others
`(22-24). Efficient transient expression of humanized variants
`in nonmyelomacells allowed us to rapidly investigate the
`relationship between binding affinity for p1854=? ECD and
`antiproliferative activity against p185#=®? overexpressingcar-
`cinomacells.
`
`MATERIALS AND METHODS
`
`Cloning of V Region Genes. The mumAb4D5 Vy and V,
`genes wereisolated by PCR amplification of mRNAfrom the
`corresponding hybridoma (6) as described (25). N-terminal
`sequencing of mumAb4D5V,and Vy was used to design the
`sense-strand PCR primers, whereasthe anti-sense PCR prim-
`ers were based on consensus sequences of murine FRresi-
`
`Abbreviations: mumAb4DS5and humAb4DS, murine and humanized
`versions of the monoclonal antibody 4DS5, respectively; ECD, ex-
`tracellular domain; ADCC, antibody-dependent cellular cytotoxic-
`ity; CDC, complement-dependent cytotoxicity; CDR, complemen-
`tarity-determining region; FR, framework region; Vy and V_, vari-
`able heavy and light domains, respectively; C region, constant
`region; V region, variable region.
`
`4285
`
`(cid:43)(cid:82)(cid:86)(cid:83)(cid:76)(cid:85)(cid:68)(cid:3)(cid:89)(cid:17)(cid:3)(cid:42)(cid:72)(cid:81)(cid:72)(cid:81)(cid:87)(cid:72)(cid:70)(cid:75)(cid:3)
`Hospira v. Genentech
`(cid:44)(cid:51)(cid:53)(cid:21)(cid:19)(cid:20)(cid:26)(cid:16)(cid:19)(cid:19)(cid:27)(cid:19)(cid:24)(cid:3)
`IPR2017-00805
`(cid:42)(cid:72)(cid:81)(cid:72)(cid:81)(cid:87)(cid:72)(cid:70)(cid:75)(cid:3)(cid:40)(cid:91)(cid:75)(cid:76)(cid:69)(cid:76)(cid:87)(cid:3)(cid:21)(cid:19)(cid:24)(cid:26)
`Genentech Exhibit 2057
`
`

`

`4286
`
`Immunology: Carteretal.
`
`Proc. Natl. Acad. Sci. USA 89 (1992)
`
`muMAb4D5 V_
`40
`30
`20
`10
`P
`s ITC KASQODVNTAVAW Y QQ K
`DIVM TQ Ss HK FMS TS VGODRV
`\TGTGAATACTGCTGTAGCCTGGTA‘\TCAACAGAAACCA.
`*
`+e ee ee
`*
`eS
`<
`eet
`GCTGATATCGTGATGACCCAGTCCCA\
`\CCTCTGTGGGCGA‘
`GCTGATATCCAGATGA’
`
`
`\CCCAGTCCCCGAGCTCCCTGTCCGCCTCTIGTGGGCGATAGGGTCACCATCACCTGCCGTGCCAGTCAGGATGTGAATACTGCTGTAGCCTGGTATCAACA\
`AGAAACCA
`Q K
`P
`DIQOMTQS PSSLUSASVGODORVTIT C_RAS Q DVN TA VAW Y Q
`Vp-CDR1
`huMAb4D5-5 V_
`Fic. 1. Nucleotide and amino
`70
`#
`#
`60
`50
`GTDFTFTIs Ss VQ
`0 acid sequences of mumAb4DS and
`GHS PKLLIY¥SASFRYTGVPODRFTGNR
`\TAGATCTGGGACGGATTTCACTTTCACCATCAGCA.
`**
`* *
`+e
`eee
`eek ee
`*
`humAb4DS-5 V, (A) and Vy (B)
`\CTACTGA‘
`\TTTACTCGGCA*
`\GTOCCTTCTICGCTICTCTGGATCCAGATCTGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAA.
`(numbered according to ref. 26).
`GKAPKLLIYS AS FLESGVPSRFSGSRSGTDFTLTIS S LQ PE
`The CDRresidues according to a
`Vp-CDR2
`sequence definition (26) and a
`structural definition (22) are un-
`derlined and overlined, respec-
`tively. The 5’ and 3’ ends of the
`oligonucleotides used for gene
`conversion mutagenesis are
`shown by arrows and mismatches
`30
`20
`10
`muMAb4D5 Vy
`eveLaeesGPELVKPGASLKLSCTASGFNIK ODT Y¥IHWV K
`between genes are shownbyas-
`\GCTTCTGGCTTCAACA’
`'ATATACACTGGGTGAAA,
`GCGTACGCTCAK
`oe
`ke
`*
`*
`ee
`*
`*
`=
`+e *
`’ACAC:
`aaa —_terisks. The asparagine-linked gly-
`GOGTA\
`CATTAAAGACACCTATATACACTGGGTGCGT==cgsylation site (#) in mumAb4D5
`EVQLUVESGGGLVQPGGSLRLSCAAS GFNIK_DT¥ IH WV R
`Vy-CDR1
`V, is used in some mumAb4D5
`molecules derived from the corre-
`sponding hybridoma. However,
`mumAb4DS5 variants, which are
`glycosylated or aglycosylated in
`VL, are indistinguishable in their
`110
`c
`100 a
`c
`a
`80
`binding affinity for the p1854ER2
`MDYWGQGaASVTVS S85
`YLQvSsS R LTS EDTAVY¥Y¥Y¥CSERWGGODGF Y
`\TGGACTACTGGGGTCAAGGAGCCTCGGTCACCGTCTCCTCG
`ECDandin their antiproliferative
`‘Ti
`ATTATIGTTCIAGATGGGGAGGGGAC
`+28
`*
`ee
`part
`pos
`eee ee
`ATGCTATGGA(
`\CGTGTGGGGTCAAGGAACCCTGGTCA\
`\TGAACA( \TGGGGAGGGGACGGCTTCT,
`
`activity with SK-BR-3 cells (C.K.,
`vVvwoeagogcTttuLuvtgrtvys
`s
`YLQMNSLRAEDTAV YY C S R_WGGD GF Y AM D
`M. Spellman, and B. Hutchins,
`Vy-CDR3
`unpublished data).
`
`huMAb4D65-5 Vy
`70
`60
`a
`50
`40
`PK FQODKATITAODTSSNTA\CACAGCC
`QRPEQGLEWIGRIY¥Y PTNGYTR Y D
`\GACACA"
`\TTTATCCTACGAATGGTTATACTAGATA’
`+28 REE
`+ eee Ree
`><
`‘+ eee
`+e
`eee
`=>
`‘AAGGGCCTGGAATGGGTTGCAAGGATTTATCCTACGAATGGTTATACTAGATATGCCGA’
`CAGGCCCOGGGT)
`\TAGCGTCAA\
`\TCCAAAAACACAGCC
`QAPGKGULEWVA _R IY PTN GY TR YAODSVKGRFTISADTS KNTA
`Vy-CDR2
`
`bA
`
`dues (25, 26) incorporating restriction sites for directional
`cloning shownbyunderlining andlisted after the sequences:
`Vi sense, 5’-TCCGATATCCAGCTGACCCAGTCTCCA-3’
`EcoRV; V, antisense, 5'-GTTTGATCTCCAGCTTGG-
`TACCHSCDCCGAA-3’ Asp718; Vy sense, 5'’-AGGTSM-
`ARCTGCAGSAGTCWGG-3’' Pst I; Vy antisense, S5’-
`TGAGGAGACGGTGACCGTGGTCCCTTGGCCCCAG-3’
`BstEII; where H is A, C, or T; S is C or G; Dis A, G, or T;
`Mis AorC; Ris A orG; Wis A or T. The PCR products were
`cloned into pUC119 (27) and five clones for each V domain
`were sequencedbythe dideoxynucleotide chain-termination
`method (28).
`Molecular Modeling. Models of mumAb4DS5 Vy and V,
`domains were constructed by using seven Fab crystal struc-
`tures from the Brookhaven Protein Data Bank(entries 2FB4,
`2RHE, 2MCP, 3FAB, 1FBJ, 2HFL, and 1RED) (29). Vy and
`V of each structure were superimposed on 2FB4 by using
`main-chain atom coordinates (INSIGHT program, Biosym
`Technologies, San Diego). The distances from each 2FB4 Ca
`to the analogous Ca in each of the superimposed structures
`wascalculated. For residues with all Ca—Ca distances <1A,
`the average coordinates for individual N, Ca, C, O, and CB
`atoms were calculated and then corrected for resultant de-
`viations from standard bond geometry by 50 cycles of energy
`minimization (DISCOVER program, Biosym Technologies) us-
`ing the AMBERforcefield (30) and fixed Ca atoms. Side chains
`of FR residues were then incorporated, followed by inclusion
`of five of the six CDR loops (except Vy—CDR3) using
`tabulations of CDR conformations(23) as a guide. Side-chain
`conformations were chosen on the basis of Fab crystal
`structures, rotamer libraries (31), and packing consider-
`ations. Three possible conformations of Vy-CDR3 were
`taken from a search ofsimilar sized loops in the Brookhaven
`Protein Data Bank or were modeled by using packing and
`solvent exposure considerations. Models were then sub-
`jected to 5000 cycles of energy minimization.
`A modelof the humAb4DS wasgenerated by using consen-
`sus sequences derived from the most abundant human sub-
`classes—namely, V; x subgroup I and Vj; subgroupIII (26).
`The six CDRswere transferred from the mumAb4DS model
`onto a human Fab model. All humAb4DS variants contain
`
`humanreplacements of mumAb4DSresiduesatthree positions
`within CDRsas defined by sequence variability (26) but not as
`defined by structural variability (22): V_-CDR1 K24R, V,-
`CDR2 RS54L and V,;-CDR2 T56S.1 Differences between
`mumAb4DSand the human consensus FRresidues (Fig. 1)
`were individually modeled to investigate their possible influ-
`ence on CDR conformation and/or binding to p1854£®2 ECD.
`Construction of Chimeric Genes. Genes encoding the chi-
`meric mAb4DSlight and heavy chains were separately as-
`sembled in previously described phagemid vectors contain-
`ing the human cytomegalovirus enhancerand promoter,a 5’
`intron, and simian virus 40 polyadenylylation signal (32).
`Briefly, gene segments encoding mumAb4DS V, (Fig. 1A)
`and REI human x,light-chain C,, (33) were precisely joined
`as were genes for mumAb4DS5 Vy (Fig. 1B) and human IgG1
`C region (34) by subcloning (35) and site-directed mutagen-
`esis as described (36). The IgG1 isotype was chosen, asitis
`the preferred humanisotype for supporting ADCC and CDC
`by using matched sets of chimeric (15) or humanizedanti-
`bodies (17). The PCR-generated V; and Vy, fragments (Fig.
`1) were subsequently mutagenized so that they faithfully
`represent the sequence of mumAb4DS determined at the
`protein level: Vy, QIE; V_, V104L and T109A. The human
`IgG1 C regionsare identical to those reported (37) except for
`the mutations E359D and M361L (Eu numbering; ref. 26),
`whichweinstalled to convert the antibody from the naturally
`rare A allotype to the much more common non-A allotype
`(26). This was an attempt to reduce the risk of anti-allotype
`antibodies interfering with therapy.
`Construction of Humanized Genes. Genes encoding chi-
`meric mAb4DSlight-chain and heavy-chain Fd fragment (Vy
`and Cy1 domains) were subcloned together into pUC119 (27)
`to create pAK1 and were simultaneously humanized in a
`single step (43). Briefly, sets of six contiguous oligonucleo-
`tides were designed to humanize Vy and V, (Fig. 1). These
`oligonucleotides are 28-83 nucleotides long, contain 0-19
`mismatchesto the murine antibody template, and are con-
`
`Wariants are denoted by the amino acid residue and number
`followed by the replacement aminoacid.
`
`#s
`
`100
`90
`DLAVYYcQQHYTTPPTFGGGTKVETI XK
`GACCTGGCAGTTTATTACTGTCAGCAACATTATACTACTCCTICCCACGTTCGGAGGGGGTACCAAGGTGGAGATCAAA
`ee
`ee
`aes
`+e
`ky
`GACTTCGCAACTTATTACIGTCAGCAACATTATACTACTCCTICCCACGTTCGGACAGGGTACCAAGGTGGAGATCAAA
`DFATYYcCQQHY TTPPTFGQGTKVETI K
`Vy-CDR3
`
`A
`
`B
`
`

`

`Immunology: Carteret al.
`
`Proc. Natl. Acad. Sci. USA 89 (1992)
`
`4287
`
`strained to have8 or 9 perfectly matched residues at each end
`to promote efficient annealing andligation of adjacent oligo-
`nucleotides. The sets of Vy and V, humanization oligonu-
`cleotides (5 pmol each) were phosphorylated with either ATP
`or [y-°2P]ATP(36) and separately annealed with 3.7 pmol of
`pAK1 template in 40 yl of 10 mM Tris-HCI (pH 8.0) and 10
`mM MgCl, by cooling from 100°C to ~20°C over ~20 min.
`The annealed oligonucleotides were joined by incubation
`with T4 DNAligase (12 units; New England Biolabs) in the
`presenceof 2 ul of 5SmM ATP and 2 ul of 0.1 M dithiothreitol
`for 10 min at 14°C. After electrophoresis on a 6% acrylamide
`sequencinggel, the assembled oligonucleotides were located
`by autoradiography and recovered by electroelution. The
`assembled oligonucleotides (~0.3 pmol each) were simulta-
`neously annealed to 0.15 pmol of single-stranded deoxyuri-
`dine-containing pAK1prepared as described (38)in 10 pl of
`40 mM Tris-HCl (pH 7.5) and 16 mM MgCl, as described
`above. Heteroduplex DNAwasconstructed by extending the
`primers with T7 DNA polymerase and transformed into
`Escherichia coli BMH 71-18 mutL as described (36). The
`resultant phagemid DNA poolwasenrichedfirst for human
`V,byrestriction purification using Xho I and then for human
`Vy byrestriction selection using Stu I as described (36, 39).
`Resultant clones containing both human V, and human Vy
`genes were identified by nucleotide sequencing (28) and
`designated pAK2. Additional humanized variants were gen-
`erated by site-directed mutagenesis (36). The mumAb4DS5 V;_
`and Vy gene segmentsin the transient expression vectors
`described above were then precisely replaced with their
`humanized versions.
`Expression and Purification of mAb4D5 Variants. Appro-
`priate mAb4DS5light- and heavy-chain cDNA expression
`vectors were cotransfected into adenovirus-transformed hu-
`man embryonic kidney cell line 293 by a high-efficiency
`procedure (32). Media were harvested daily for up to 5 days
`and the cells were refed with serum-free medium. Antibodies
`were recovered from the media and affinity purified on
`protein A-Sepharose CL-4B (Pharmacia) as described by the
`manufacturer. The eluted antibody was buffer-exchanged
`into phosphate-buffered saline by G25 gelfiltration, concen-
`trated by ultrafiltration (Amicon), sterile-filtered, and stored
`at 4°C. The concentration of antibody was determined by
`both total IgG and antigen binding ELISAs. The standard
`used was humAb4D5-5, whose concentration had been de-
`termined by amino acid composition analysis.
`Cell Proliferation Assay. The effect of mAb4DSvariants on
`proliferation of the human mammary adenocarcinomacell
`line SK-BR-3 was investigated as described (6) by using
`saturating mAb4DSconcentrations.
`
`Affinity Measurements. mAb4D5 variant antibodies and
`p185#=®2 ECD werepreparedas described(40) and incubated
`in solution until equilibrium was found to be reached. The
`concentration of free antibody was then determined by
`ELISA using immobilized p1854=®? ECD and was used to
`calculate affinity (Kg) as described (41). The solution-phase
`equilibrium between p1854=®2 ECD and mAb4DS variants
`was found notto be grossly perturbed during the immobilized
`ECD ELISA measurementoffree antibody.
`
`RESULTS
`
`Humanization of mumAb4D5. The mumAb4DS5 V, and Vy
`gene segmentswere first cloned by PCR and sequenced(Fig. 1).
`The V region genes were then simultaneously humanized by
`gene conversion mutagenesis using preassembled oligonucleo-
`tides. A 311-mer oligonucleotide containing 39 mismatches to
`the template directed 24 simultaneous amino acid changes
`required to humanize mumAb4DS5 V,. Humanization of
`mumAb4DS Vy required 32 amino acid changes, which were
`installed with a 361-mer containing 59 mismatches to the
`mumAb4DStemplate. Twoofeight clones sequencedprecisely
`encode humAb4D5-5, although oneof these clones contained a
`single nucleotide imperfection. The six other clones were es-
`sentially humanized but contained a small numberof errors: <3
`nucleotide changes and <1 single nucleotide deletion perkilo-
`base. Additional humanizedvariants (Table 1) were constructed
`by site-directed mutagenesis of humAb4DS-5.
`Expression levels of humAb4DSvariants were 7-15 ng/ml
`as judged by ELISA using immobilized p185"£®"2 ECD.
`Successive harvestsof five 10-cm plates allowed 200-500 ug
`of each variant to be produced in a week. Antibodiesaffinity
`purified on protein A gave a single band on a Coomassie
`blue-stained SDS/polyacrylamide gel of mobility consistent
`with the expected mass of ~150 kDa. Electrophoresis under
`reducing conditions gave two bands consistent with the
`expected mass of free heavy (48 kDa) andlight (23 kDa)
`chains (data not shown). N-terminal sequenceanalysis (10
`cycles) gave the mixed sequence expected (see Fig. 1) from
`an equimolar combination of light and heavy chains.
`humAb4D5Variants. In general, FR residues were chosen
`from consensus human sequences (26) and CDRresidues
`were chosen from mumAb4DS5. Additional variants were
`constructed by replacing selected human residues in
`humAb4D5-1 with their mumAb4DScounterparts. These are
`Vu residues 71, 73, 78, 93, plus 102 and V, residues 55 plus
`66. Vy residue 71 has previously been proposed byothers
`(24) to be critical to the conformation of Vy—-CDR2. Amino
`acid sequencedifferences between humAb4D%Svariant mol-
`ecules are shownin Table 1 together with their p1854#=8? ECD
`
`Table 1.
`
`mAb4DS5
`variant
`humAb4DS5-1
`humAb4D5-2
`humAb4DS-3
`humAb4D5-4
`humAb4D5-5
`humAb4DS5-6
`humAb4DS-7
`humAb4DS5-8
`humAb4D5
`
`71
`(FR3)
`R
`Ala
`Ala
`Ala
`Ala
`Ala
`Ala
`Ala
`Ala
`
`73
`(FR3)
`D
`D
`Thr
`Thr
`Thr
`Thr
`Thr
`Thr
`Thr
`
`p1854ER2 ECDbindingaffinity and anti-proliferative activities of mAb4D5 variants
`Vu residue
`V{ residue
`78
`(FR3)
`L
`L
`Ala
`L
`Ala
`Ala
`Ala
`Ala
`Ala
`
`Relative cell
`Ka,
`66
`55
`102
`proliferation
`nM
`(FR3)
`(CDR2)
`(CDR3)
`102
`25
`G
`E
`Vv
`101
`4.7
`G
`E
`Vv
`66
`4.4
`G
`E
`Vv
`56
`0.82
`Arg
`E
`Vv
`48
`11
`Arg
`E
`Vv
`51
`0.22
`Arg
`Tyr
`Vv
`53
`0.62
`Arg
`E
`Tyr
`54
`0.10
`Arg
`Tyr
`Tyr
`37
`0.30
`Arg
`Tyr
`Tyr
`Human and murineresidues are shownin one-letter and three-letter amino acid codes, respectively. Kg values for the p1854=®2 ECD were
`determined by the method of Friguet etal. (41) and the standard error of each estimate is +10%. Proliferation of SK-BR-3 cells incubated for
`96 hr with mAb4DSvariants is shownas a percentage of the untreated control as described (7). Data represent the maximal antiproliferative
`effect for each variant (see Fig. 2) calculated as the meanoftriplicate determinations at a mAb4D5 concentration of 8 ug/ml. Data are all taken
`from the same experimentand the estimated standard error is +15%.
`
`|
`
`93
`(FR3)
`A
`A
`Ser
`Ser
`Ser
`Ser
`Ser
`Ser
`Ser
`
`

`

`4288
`
`Immunology:Carteretal.
`
`
`proliferation
`
`Percentofcontrolcell
`
`huMAb4D5-8
`
`4
`
`12
`8
`[MAb4DSvariant] jig/ml
`
`16
`
`Proc. Natl. Acad. Sci. USA 89 (1992)
`
`placementofR71 in humAb4D5-1 with the corresponding murine
`residue, A71 (humAb4DS5-2). In contrast, replacing Vz L78 in
`humAb4DS-4 with the murine residue A78 (humAb4D5-5) does
`not significantly change theaffinity for the p185#=®2 ECD or
`changeantiproliferative activity, suggesting that residue 78 is not
`of critical functional significance to humAb4D5 in interacting
`with p185HER2 ECD.
`Vi,residue 66 is usually a glycine in human and murine
`«-chain sequences (26) but an arginine occupies this position
`in the mumAb4D5« light chain. The side chain of residue 66
`is likely to affect the conformation of V;-CDR1 and V,-
`CDR2 andthe hairpin turn at residues 68-69 (Fig. 3). Con-
`sistent with the importanceofthis residue, the mutation V;_
`G66R (humAb4DS5-3 — humAb4D5-5)increases the affinity
`for the p1854=®2 ECD by4-fold with a concomitant increase
`in antiproliferative activity.
`From molecular modeling, it appears that the side chain of
`mumAb4DS5 V, Y55 mayeitherstabilize the conformation of
`Vu-CDR3 or provide aninteraction at the Vj—Vy interface.
`The latter function may be dependenton the presence of Viz
`Y102. In the context of humAb4D5-5 the mutations V;, ESSY
`(humAb4DS5-6) and Vy V102Y (humAb4DS5-7) individually
`increase the affinity for p1854=®2 ECD by 5-fold and 2-fold,
`respectively, whereas together (humAb4D5-8) they increase
`the affinity by 11-fold. This is consistent with either proposed
`role of Vi Y55 and Vy Y102.
`Immune Function of humAb4D5-8. humAb4D5-8
`efficiently mediates ADCC against SK-BR-3 breast carcinoma
`cells, which overexpress p1854=®? at high levels as anticipated
`from its IgG1 isotype (Table 2). In contrast, humAb4D5-8 is
`very inefficient in mediating ADCCagainst the normal lung
`epithelium cell line WI-38, which expresses p185#=F2 at 100-
`fold lower levels than SK-BR-3 cells (Table 2). The murine
`parent antibodyis notvery effective in mediating ADCCagainst
`either SK-BR-3 or WI-38 cells.
`
`DISCUSSION
`
`mumAb4DSis potentially useful for human therapysince it is
`cytostatic toward human breast and ovarian tumorlines over-
`expressing p1854=®2, Here we have humanized mumAb4DSin
`an attemptto improveits potential clinical efficacy by reducing
`its immunogenicity andtailoring the Fc region to support ADCC
`and possibly CDC.
`Rapid humanization of humAb4D5was facilitated by the
`gene conversion mutagenesis strategy developed here using
`long preassembled oligonucleotides. This method uses less
`
`Fic. 3. Stereoview of a-car-
`bon tracing for model of hum-
`Ab4DS5-8 V, and Vy. The CDR
`residues (26) are shown in boldface
`and side chains of Vy; residues
`A71, T73, A78, S93, and Y102 and
`V_ residues Y55 and R66 (see Ta-
`ble 1) are shown.
`
`Inhibition of SK-BR-3 proliferation by mAb4D5variants.
`Fic. 2.
`Relative cell proliferation was determined as described (7) and data
`(average oftriplicate determinations) are presented as a percentage
`ofresults with untreated cultures for mumAb4D5, humAb4D5-8, and
`humAb4DS5-1.
`
`binding affinity and maximal antiproliferative activities
`against SK-BR-3cells. Very similar Ka values were obtained
`for binding mAb4D5variants to either SK-BR-3 cells (C.K.
`and N. Dua, unpublisheddata) or to p185#£®? ECD(Table1).
`The most potent humanizedvariant designed by molecular
`modeling, humAb4D5-8, contains five FR residues from
`mumAb4D5. This antibody binds the p185#=®2 ECD 3-fold
`more tightly than does mumAb4DSitself (Table 1) and has
`comparable antiproliferative activity with SK-BR-3 cells
`(Fig. 2). In contrast, humAb4D5-1 is the most humanized but
`least potent mumAb4DSvariant,created by simplyinstalling
`the mumAb4DS5 CDRsinto the consensus human sequences.
`humAb4DS5-1 binds the p1854=®? ECD 80-fold less tightly
`than does the murine antibody and hasno detectableantipro-
`liferative activity at the highest antibody concentration in-
`vestigated (16 ug/ml).
`The antiproliferative activity of humAb4D5 variants
`against p1854=®? overexpressing SK-BR-3cells is not simply
`correlated with their bindingaffinity for the p185"=22 ECD—
`e.g., installation of three murine residues into the Vy domain
`of humAb4DS5-2 (D73T, L78A, and A93S) to create
`humAb4D5-3 does not changethe antigenbindingaffinity but
`does confersignificant antiproliferative activity (Table 1).
`The importance of Vy residue 71 (24) is supported by the
`
`observed 5-fold increase in affinity for p1854=82 ECD onre-
`
`

`

`Immunology: Carteret al.
`
`Proc. Natl. Acad. Sci. USA 89 (1992)
`
`4289
`
`25:1
`12.5:1
`6.25:1
`3:13:1
`
`25:1
`12.5:1
`6.25:1
`3.13:1
`
`Antibody concentration, 100 ng/ml
`<1.0
`9.3
`7.5
`<1.0
`11.1
`4.7
`<1.0
`8.9
`0.9
`<1.0
`8.5
`4.6
`Antibody concentration, 10 ng/ml
`<1.0
`3.1
`6.1
`<1.0
`1.7
`5.5
`13
`2.2
`2.0
`<1.0
`0.8
`2.4
`
`40.6
`36.8
`35.2
`19.6
`
`33.4
`26.2
`21.0
`13.4
`
`10.
`
`11.
`
`12.
`
`13.
`
`14.
`
`16.
`
`17.
`
`18.
`19.
`
`20.
`
`21.
`
`22.
`23.
`
`24.
`
`25.
`
`26.
`
`21
`
`29.
`
`31.
`32.
`
`33.
`
`35.
`
`36.
`
`37.
`
`38.
`
`39.
`
`41.
`
`42.
`
`43.
`
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`Sensitivity to ADCC of human cell lines WI-38 (normal lung
`epithelium) and SK-BR-3 (breast tumor), which express 0.6 and 64
`pg of p185HER2 per yg ofcell protein, respectively, as determined by
`ELISA (40). ADCCassays were carried out as described (15) using
`interleukin 2 activated human peripheral blood mononuclear cells as
`effector cells and either WI-38 or SK-BR-3 target cells in 96-well
`microtiter plates for 4 hr at 37°C at different antibody concentrations.
`Values given represent percentage specific cell lysis as determined
`by *!Cr release. The estimated standard error in these quadruplicate
`determinations was +10%.
`
`than half the amount of synthetic DNA, as does total gene
`synthesis, and does not require convenientrestrictionsites in
`the target DNA. Our method appearsto be simpler and more
`reliable than a similar protocol recently reported (42). Tran-
`sient expression of humAb4DS5 in human embryonic kidney
`293 cells permitted the isolation of 0.2- to 0.5-mg humAb4D5
`variants for rapid characterization by growth inhibition and
`antigen binding affinity assays. Furthermore, different com-
`binations of light and heavy chain were readily tested by
`cotransfection of corresponding cDNA expression vectors.
`The crucial role of molecular modeling in the humanization
`of mumAb4DS is illustrated by the designed variant
`humAb4D5-8, which binds the p185#=®2 ECD 250-fold more
`tightly than the simple CDR loop swapvariant humAb4D5-1.
`It has previously been shownthat the antigen bindingaffinity
`of a humanized antibody can be increased by mutagenesis
`based on molecular modeling (17, 20). Here we have designed
`a humanized antibody that binds its antigen 3-fold more
`tightly than the parent antibody andis almost as potent in
`blocking the proliferation of SK-BR-3 cells. While this result
`is gratifying, assessmentof the success of molecular model-
`ing must await the outcomeof ongoing x-ray crystallographic
`structure determination.
`humAb4D5-8 also supports cytotoxicity via ADCC against
`SK-BR-3 tumorcells in the presence ofhuman effectorcells but
`is not effective in directing the killing of normal (WI-38)cells,
`which express p1854E®? a