Pfizer v. Genentech
`IPR201(cid:26)-01488
`Genentech Exhibit 202(cid:23)
`
`

`

`4182
`
`Immunology: Gormanetal.
`
`Proc. Natl. Acad. Sci. USA 88 (1991)
`
`were CDR-grafted (9) into the 1.5-kb HindIII fragment from
`pNH316 encoding the Campath-1H antibody heavy chain by
`in vitro mutagenesis with three oligonucleotides.
`The KOL-based reshaped Vy region was created by in
`vitro mutagenesis of a Campath-9 antibody Vy region cDNA
`clone in M13-VHPCRI1with five oligonucleotides that were
`designed to mutate the Campath-9 Vy region framework
`residues into the corresponding residues of the KOL anti-
`body (18). These five mutagenic oligonucleotides were si-
`multaneously introduced in a single mutagenesis reaction.
`Twelve clones were sequenced and each clone had incorpo-
`rated the five mutagenic oligonucleotides. The plasmid
`pVyKOL/Cg: encoding the KOL-based reshaped heavy
`chain consists of the following adjacently ligated fragments:
`the 9.8-kb vector pHBAPr-1-gpt (14) linearized atits cloning
`site with HindIII and BamHIcontaining the B-actin pro-
`moter, xanthine-guanine phosphoribosyltransferase, and am-
`picillin-resistance genes; the 39-bp HindIII—Nco I linker; a
`698-bp Nco I-BamHI fragment encoding the KOL-based
`reshaped Vy region; a 2.3-kb BamHI-Sph I fragment con-
`taining a human Cg;region gene(15); and the 20-bp Sph I-Bgl
`II synthetic linker.
`The plasmid pV,REI/C,, encoding the reshapedlight chain
`is identical to the plasmid pLD9 (16) except that the three
`CDRs of the Campath-9 antibody V, region were CDR-
`grafted (9) into the 748-bp HindIII fragment from pLD9
`encoding the Campath-1H antibody light chain by in vitro
`mutagenesis with three oligonucleotides.
`Transfections and Antibody Purification. The CD4-
`expressing cell line HCD4-NB2is a cloneofthe rat T-cell line
`NB2-6TG stably transfected by electroporation with the
`expression vector pSFSVneo(19) containing cDNA encod-
`ing the human CD4 antigen (20).
`Plasmids encoding antibody chains were cotransfected as
`described (21) into dihydrofolate reductase-deficient Chinese
`hamster ovary cells (10° cells per 75-cm? flask) using 9 wg and
`1 yg of the appropriate heavy and light chain constructs,
`respectively. Transfectants were selected in medium con-
`taining 5% dialyzed fetal bovine serum for 2-3 weeks, and
`antibody-secreting clones were identified by ELISA ofcul-
`ture supernatants. Chimeric and reshaped antibodies were
`purified from culture supernatants using protein A-Sepharose
`CL-4B (Pharmacia) column chromatography as described
`(22). Antibody concentrations were determined by absor-
`bance at 280 nm.
`Immunofluorescence and Flow Cytometry. HCD4-NB2
`cells were washed with staining medium (phosphate-buffered
`saline containing 0.1% bovine serum albumin, 1% heat-
`inactivated normal rabbit serum, and 0.1% sodium azide) and
`then incubated with either the chimeric or reshaped antibod-
`ies (10° cells per 0.1 ml) diluted in staining medium for 1 hr
`at 4°C. The cells were washed and then incubated with
`fluorescein isothiocyanate-conjugated anti-human IgG] (+
`chain-specific) antibodies (The Binding Site, Birmingham,
`U.K.) diluted 1:30 in staining medium for 1 hr at 4°C.
`Propidium iodide (100 g/ml final concentration) was added
`during the last 10 min of incubation. Cells were thoroughly
`washed andresuspendedin 0.5 ml of staining medium. Mean
`cellular fluorescence (3000 live cells per sample) was deter-
`mined with a Cytofluorograph (model 50-H Ortho Instru-
`ments). Propidium iodide-stained dead cells were gated-out.
`Fifty percent antigen bindingtiters were determinedbyfitting
`the data to a sigmoid curve by a least squares iterative
`procedure (23).
`Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC).
`Antibodies were assayed by ADCC with activated human
`peripheral blood mononuclear cells (24). Briefly, 5 x 104
`HCD4-NB2cells were labeled with *#Cr and incubated for 1
`hr at room temperature with different concentrations of
`antibodies. A 75-fold excess of activated cells was added as
`
`effectors. After 4 hr at 37°C, cell death was determined by
`measuring *!Crrelease.
`
`RESULTS
`
`Cloning of V, and Vy Region cDNA. cDNAsencoding the
`V. and Vy regions from the Campath-9 antibody-secreting
`clone YNB46.1.8SG2B1.19 (10) were isolated by PCR using
`primers that amplify the segment of cDNA encoding the
`amino-terminal region through the joining region (11). Vr.
`region clones were first screened by hybridization with a
`32P-labeled oligonucleotide probe complementary to CDR 2
`of the light chain expressed by the rat Y3-Agl.2.3 myeloma
`cell line (12) that was used as the fusion partner to generate
`the Campath-9 antibody-secreting hybridoma. Subsequent
`nucleotide sequenceanalysis wasrestricted to clones that did
`not contain sequence complementary to this probe (about 5%
`of clones). In this manner, two cDNAclonesfrom indepen-
`dent PCR amplifications were identified that encoded iden-
`tical V, regions. Nucleotide sequence analysis of random Vz
`region clones from two independent PCR amplifications
`revealed a single species of Vy region cDNA. These cDNA
`sequences have been submitted to the GenBank data base,
`and their predicted amino acid sequencesare shown(Fig. 1).
`As no additional Vi or Vy region-encoding clones were
`identified, it was assumed that these sequences were derived
`from the Campath-9 antibody genes.
`Chimeric Antibody Constructs. Plasmids were constructed
`that encoded a rat/human chimeric version of the Campath-9
`antibody. The plasmid pVyrat/Cg) encodes a chimeric heavy
`chain consisting of the Campath-9 Vy region (Fig. 1A) and a
`human Cg, region. The plasmid pV,rat/C, encodes a chi-
`meric light chain consisting of the Campath-9 V; region (Fig.
`1B) and a human C,. These chimeric heavy andlight chains
`were coexpressed in Chinese hamsterovary cells to produce
`a chimeric antibody.
`Reshaped Antibody Heavy Chain Constructs. Possibly the
`largest unknown variable whenreshaping an antibodyis the
`selection of the human immunoglobulin V region from which
`the framework sequences are derived. Because the frame-
`work regions hold the CDRsin their correct spatial orienta-
`tion and can sometimes even participate in antigen binding
`(29), this selection could be important. At present, there are
`insufficient published reshaping results to generalize a ‘‘best
`framework’’ selection strategy. Reshaping experiments to
`date (9, 30-32) have not compared the effectiveness of
`different human frameworks incorporating the same rodent
`CDRs.
`To investigate the importance of frameworkselection and
`to maximize our chancesof producing a functional reshaped
`CD4 antibody, we have designed two different versions of
`reshaped Vy regions. In the first case, we designed a re-
`shaped Vy region that derives its CDRs from the Campath-9
`Vu region andits framework sequences from the NEW-based
`framework that had been used previously for the reshaped
`antibody Campath-1H (9) and others (30, 31). Given the
`demonstrable antigen binding of these antibodies, it was
`reasonable to try the same framework sequences as well. A
`plasmid was thus constructed, pVyNEW/Cg, that encodes
`a reshaped heavy chain consisting of an NEW-based Vy
`region with Campath-9 Vy region CDRs (Fig. 1A) and a
`human Cg;region.
`In the secondcase, we designed a reshaped Vy region that
`derives its CDRs from the Campath-9 Vy region and its
`framework sequences from the Vy region of the human
`myeloma protein KOL (18). The Vy region of KOL was
`chosen becauseofall known human heavychain V regionsits
`overall amino acid sequence is very homologous to the
`Campath-9 Vy region (Fig. 1A) containing 72% identical
`residues (excluding gaps introduced for alignment purposes).
`
`

`

`Immunology: Gormanetal.
`
`Proc. Natl. Acad. Sci. USA 88 (1991)
`
`4183
`
`A
`
`CAMPATH-9
`NEW
`CAMPATH-1H
`NEW-based resh.
`KOL
`KOL-based resh.
`
`CAMPATH-9
`NEW
`CAMPATH-1H
`NEW-based resh.
`KOL
`KOL-based resh.
`
`B
`
`CAMPATH-9
`REI
`CAMPATH-1H
`REI-based resh.
`
`CAMPATH-9
`REI
`CAMPATH-1H
`REI-based resh.
`
`70
`60
`50
`40
`30
`20
`10
`QVQLQESGGG LVQPGRSLKL SCAASGLTFS NYGMAWVRQA PTKGLEWVAT ISHDGSD--T YFRDSVKGRF
`...-EQ..P.
`..R.SQT.S. T.TV
`.
`.DYYT....P .GR....IGY VFYH.TSDD.
`---TPLRS.V
`weeeeeeeP.
`..R.SQT.S. T.TV..F..T DFY.N....P .GR....IGF .RDKAKGYT. EYNP..... Vv
`woes oecePs <eReSQTeSs TeTVeeFicl cus s www ss P sGRewsedGe wees ome Sin,
`wie reyes 9 xe Vv
`osska & Vis 3 38 Visewsss R.
`..SS..FI..
`S.A.Y
`Ges
`I
`.WD
`Qn HYA cess cnx
`aod Veale 6% Vinseces Re ss6SicFlos sass cunwew « Givers i wrere
`sxeree orese au tt. re
`
`129
`120
`110
`100
`90
`80
`TISRDNGKST LYLQMDSLRS EDTATYYCAR QG-------- TIAG-IRHWG QGTTVIVSS
`-MLV.TS.NQ FS.RLS.VTA A...V..... N--------- L...C.DV..
`..SL.....
`-MLV.TS.NQ FS.RLS.VTA A...V..... E.H------- -T.APFDY..
`..SL.....
`-MLV.TS.NQ FS.RLS.VTA A...V..... emmane ee a ee SLs « esexe
`«e.ee-S.N.
`.F.......P ...GV.F... D.GHGFCSSA SCF.P-DY..
`... By s ays
`cece BeN. CBee ee oP oe GVeBe ce
`commmmmmm oes AUG 8s) Ws steers HOR
`
`70
`60
`50
`40
`30
`20
`10
`DIQLTQSPVS LSASLGETVN IECLASEDIY SDLAWYQQKP GKSPQLLIYN TDTLQNGVPS RFSGSGSGTQ
`.. M....S.
`....V.DR.T .T.Q..Q..1 KY.N....T.
`..A.K...-E ASN. A... cece ew eeee D
`~oeM....S.
`...-V.DR.T .T.K..QN.D KY.N......
`..A.K...-. oNN..T. ww. cece ee eee D
`oe eM... Se 20. -VUDR.T Ton ccc ee www ween oe A.Riivci eeiiwwrsee 21 seees ue D
`
`107
`100
`90
`80
`YSLKINSLQS EDVATYFCQQ YNNYPWIFGG GTKLEIK
`oTFT.S...P ..2T...¥e0.
`-QSL.Y...Q ....Q.T
`FIFT.S...P ..1...¥.L. HISR.R...Q ...V...
`FIFT.S...P ..1...Yous weveceeee Q ...V...
`
`Fic. 1. Comparison of the amino acid sequencesof the heavy (A) andlight (B) chain V regions described in the text. Dots indicate residues
`that are identical to the corresponding residue in Campath-9. Hyphens represent spaces introduced in the sequences by Gap (25) to aid the
`alignment. CDRs of Campath-9 are underlined and residues encoded within the amplification primers and cloning vectors are overlined. resh.,
`Reshaped. Sequences of NEW, KOL,and REIare from the Swiss-Prot protein sequence data base, release 14. It should be noted that there
`are some minor sequencedifferences of NEW and KOLasrecordedin the various data bases—for example, Swiss-Prot and Brookhaven(26).
`The actual framework sequences of the NEW- and RE]I-based reshaped V regions described here are identical to those of the Campath-1H
`antibody (9), which differ only slightly from the reported framework sequences of NEW (27) and REI (28). For consistency, and given the
`demonstrable antigen binding of this reshaped antibody, identical framework sequences were usedhere.
`
`This was determined by a computersearch of several data
`bases. By contrast, the NEW Vy region sequencehas only
`47% identical residues. We reasoned that since the primary
`function of the framework sequenceis to hold the CDRsin
`their correct spatial orientation, we could maximize the
`chancesof retaining correct CDRstructure (and henceanti-
`genaffinity) by deriving framework sequences from a human
`Vu region that is most homologousto that of the rodent. Of
`the several homologous human Vy regions available, the
`choice of KOL was made because its three-dimensional
`structure is well characterized. A plasmid was thus con-
`structed, pVyKOL/Cg:,
`that encodes a reshaped heavy
`chain consisting of a KOL-based Vy region with Campath-9
`Vu region CDRs(Fig. 1A) and a human Cg;region.
`Reshaped Antibody Light Chain Construct. We have de-
`signed a reshaped V_,region that derives its CDRs from the
`Campath-9 V, region and its framework sequencesfrom the
`REI-based frameworkthat has been used previously for the
`reshaped antibody Campath-1H (9). Again, given the demon-
`strable antigen binding of this antibody, it was reasonable to
`try the same frameworkas well. A plasmid was constructed,
`pV.REI/C,,, that encodes a reshapedlight chain consisting of
`an RE]I-based V, region with Campath-9 V; region CDRs
`(Fig. 1B) and a human C, region. A second reshaped V,
`version was not created as with the reshaped Vy region
`because REI is already highly homologous (67% identical
`residues) to the rat Vy region of Campath-9. Thusthis
`reshaped light chain was coexpressed with the reshaped
`heavychains in Chinese hamsterovary cells to produce two
`reshaped antibodies (KOL- and NEW-based)differing only
`in their human-derived Vy region framework sequences.
`
`Properties of Chimeric and Reshaped Antibodies. The abil-
`ities of the chimeric and reshaped antibodies to bind the
`CD4* cell line HCD4-NB2 were compared by immunofluo-
`rescence staining (Fig. 2). The chimeric and KOL-based
`reshaped antibodies stained CD4* cells well. The titration
`curvesof these two antibodies werefitted to a sigmoid curve,
`and the concentrations (mean + SEM) of chimeric and
`KOL-based reshaped antibodies needed to achieve 50%
`antigen saturation were determinedto be 2.21 + 0.16 and 7.16
`+ 0.45 ug/ml, respectively. Thus the avidity of the KOL-
`based reshaped antibodyis only slightly reduced asit only
`
`800
`700
`600
`500
`400
`300
`200
`100
`
`fluorescence
`Mean
`
`Antibody concentration, ug/ml
`Fic. 2. Fluorescence of CD4* cells stained with chimeric and
`reshaped antibodies. 0, Campath-9 chimeric antibody; e, KOL-
`based reshaped antibody; A, NEW-based reshaped antibody; a,
`Campath-1H antibody (isotype-matched negative control). The
`KOL- and NEW-based reshaped antibodies have the same REI-
`based reshapedlight chain.
`
`

`

`4184
`
`Immunology: Gormanetal.
`
`Proc. Natl. Acad. Sci. USA 88 (1991)
`
`e
`
`Saft<5 01
`
`4
`5oe°—~o
`oe
`@
`A
`
`a
`
`San
`
`
`
`Percentlysis
`
`Antibody concentration, ug/ml
`
`Fic. 3. ADCC with chimeric and reshaped antibodies. Symbols
`are the sameasin Fig.2.
`
`takes three times the amountofthis antibody to give the same
`50% binding as the chimeric antibody. All KOL Vy region
`frameworkresidues were incorporated into this reshaped Vy;
`region exceptfor the proline at position 124 (Fig. 1A), which
`was kept as threonine. A second version that incorporates
`this proline residue gave similar results (not shown). By
`contrast, the NEW-based reshaped antibody stained CD4*
`cells only poorly even at the higher concentrations. The
`control Campath-1H antibody did not stain cells at any
`concentration. Also, the chimeric and KOL-based reshaped
`antibodies were effective in cell-mediated lysis, whereas the
`NEW-based reshaped and control Campath-1H antibodies
`wereineffective (Fig. 3).
`
`DISCUSSION
`
`framework sequences, KOL and NEW, and found one form
`to be far superior to the other. The KOL-based CD4 antibody
`retained biological activity in ADCCassays and had a relative
`binding avidity only slightly reduced from the unaltered V
`region sequencesof the chimeric CD4 antibody. In contrast,
`the NEW-based CD4 antibody, though still retaining speci-
`ficity for CD4, had a considerably reducedrelative binding
`avidity and had no biological activity. The KOL Vy region
`has a 72% homology to Campath-9 Vy region, whereas the
`NEWVjframework has only 47% homology. In this case
`then, it would seem that the selection of a humanVregion
`A
`1
`10
`framework that was highly homologous to the rodent V
`region was the best strategy for framework selection. We
`have also successfully reshaped a CD3 antibody by the same
`approach (E.G.R., unpublished data), so this strategy may
`prove to be generally applicable to antibody reshaping.
`Arguably, although such a bestfit strategy has been applied
`here to whole antibody V regions, it might also be applied
`separately to individual variable, diversity, and joining re-
`combining segments of an antibody V region.
`The different avidities we observe when reshaping with
`KOL- and NEW-basedframeworksarelikely to be due to the
`complex intrachain associations between CDR and frame-
`work residues. Alternatively, different interchain V region
`associations between these two heavychains and the REI-
`based reshapedlight chain mayalso play a role. One possible
`structural explanation for the differences between the KOL-
`and NEW-based reshaped antibodies is described by Tra-
`montano et al. (34). In studies of a series of solved immu-
`noglobulin structures they observedthat the conformation of
`the heavy chain CDR 2, ‘‘H2 loop,”’ is dependent upon the
`length and sequenceofthis loop andits interaction with the
`frameworkresidue at position 71 (Kabat numbering system).
`Wehave described here the successful reshaping of the CD4
`KOL and NEW havedistinctly different structures in this
`antibody Campath-9. This result, together with the previ-
`region and a different residue at position 71. The H2 loop of
`ously described reshaping of other therapeutic antibodies (9,
`the Campath-9 antibody is very similar to that of KOL and
`32), demonstratesthe feasibility of applying this concept to
`both contain an arginine residue at the relevant framework
`the many rodent-derived monoclonal antibodies with clinical
`position(this is position 74 in Fig. 1A). Perhaps the change of
`potential. The Campath-1H antibody has already been used
`this residue upon reshaping to the NEW-based framework
`successfully in clinical studies of lymphomatherapy (33) and
`accounts in part for its low avidity. However, it should be
`in the treatment of an autoimmunedisorder(10). In this later
`noted that Campath-1H was successfully reshaped to the
`case,
`the Campath-1H antibody was combined with the
`NEW-based frameworkdespite having a substantially differ-
`Campath-9 antibody to give a long-term remission in a case
`ent H2 loop length and sequence andalso a different frame-
`work residue at position 71 (34). Clearly the ability to use
`of systemic vasculitis that appeared intractable prior to
`Campath-9 antibody treatment. Although therelative contri-
`structural features like this to reliably predict a suitable
`strategy for reshaping will benefit from more examples where
`butions and importance of the Campath-1H and Campath-9
`different strategies are experimentally compared.
`antibodies cannotbe ascertained from this single case study,
`In previous studies, genes encoding reshaped antibodies
`the availability of Campath-9 as a reshaped antibody, Cam-
`were produced either by total synthesis of the desired se-
`path-9H (the KOL-basedversion), should provide for further
`useful clinical studies.
`quence(32) or byin vitro mutagenesis of a human V region
`sequence to incorporate the rodent CDRs (9, 30, 31). We
`Our reshaping of Campath-9 into Campath-9Hraises in-
`propose the following strategy based on our successful re-
`teresting questions regarding general strategies for the re-
`shaping experiments wherebythe isolated rodent V region is
`shaping of rodent antibodies. At present, there are only four
`used as a Starting point for constructing reshaped V regions.
`additional reports of reshaped antibodies and in no case has
`Here, human framework sequences are transferred to the
`the effectiveness of two different human antibody frame-
`rodent V regions by meansof in vitro mutagenesis. When
`works regions been compared (9, 30-32). Three of these
`coupled with the bestfit method from above, mutagenic
`antibodies had reshaped Vy regions based on NEW (9, 30,
`oligonucleotides can be highly homologous to the rodent
`31), two of which had reshaped V, regions based on REI (9,
`frameworks, and hencethe efficiency of mutagenesisis high.
`31), regardless of whether closer homologies existed in the
`In the case of Campath-9H, this was accomplishedinasingle
`sequencedatabases. In the fourth example, the antibody was
`mutagenesis reaction with five oligonucleotides 33-58 bases
`reshaped based on a homologous human framework,but 12
`of the 29 residuesthatdiffered between the human and mouse
`in length. This strategy should be readily applicable to any
`monoclonal antibody for which the cDNA hasbeen cloned
`Vy region frameworks were left as in the mouse sequence,
`and for which a homologous human framework can be
`and no data were presented that compared this reshaped
`identified in the sequence data bases. It is also interesting to
`antibody with a form in whichall of the framework residues
`note that expression in the nonlymphoid Chinese hamster
`were derived from the human sequence (32). For the reshap-
`ovary cell line results in an antibody with demonstrable
`ing of Campath-9, we have shownthat the selection of a
`activity in ADCC. Thecapacity ofthis cell line for high-level
`particular human framework can be importantin the reten-
`expression of reshaped antibodies (16) should facilitate the
`tion of antibody avidity. We have made two reshapedanti-
`large-scale production of this antibody for clinical studies.
`bodies that differ only in their usage of human Vy region
`
`

`

`Immunology: Gormanetal.
`
`Proc. Natl. Acad. Sci. USA 88 (1991)
`
`4185
`
`Wethankthe following for their helpful discussions and assistance:
`G. Winter and P. Jones for the vectors M13-VKPCRI1 and M13-
`VHPCRI1, J. S. Crowe for Campath-1H cDNA, M. Page for the
`plasmids pLD9 and pNH316,J. Ivanyi and J. Howardforthe cell line
`NB2-6TG, A. Lesk, and C. Chothia. We thank H. Spence for
`synthetic oligonucleotide synthesis, H. Kruger-Grayfor flow cytom-
`etry assistance, and M. Frewin for technical assistance. This work
`was supported by the Medical Research Council, United Kingdom,
`Wellcome Biotech PLC, and the Gilman Foundation. S.D.G. is a
`recipient of a Special Fellowship from the Leukemia Society of
`America.
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