0022· I 767 /9 I / I 4 712·4366$02.00/0
`'l'ff& J OURNAL OF IMMUSOLOCY
`Copyright C 1991 by The American Association or Immunologists
`
`Vol. 147. 4366-4373. No. 12. December 15. 1991
`Printed In U.S.A.
`
`CONSTRUCTION, EXPRESSION AND CHARACTERIZATION OF HUMANIZED
`ANTIBODIES DIRECTED AGAINST THE HUMAN cx/{3 T CELL RECEPTOR
`
`CLYDE W . SHE ARMAN,'* DAN POLLOCK ,* GARY WHITE,* KA THY HEHIR,*
`GORDON P. MOORE.2 * E. J . KANZY!AND ROLAND K URRL Et
`From the •Genzyme Corporation. Framingham. MA 01701: and 'Behringwerke Aktiengesellschaft. D-3550
`Marburg. West Germany
`
`Completely humanized antibodies with specificity
`for the human a/{J TCR have been produced by ge(cid:173)
`netic engineering. The L and H chain V region exons
`encoding the murine mAb BMA 031 CD regions and
`human EU framework regions were synthes ized and
`replaced into previously isolated genomic frag(cid:173)
`ments. These fragments were inserted into mam(cid:173)
`malian expression vectors containing the human 11.
`and "Y 1 C region exons. Two variants were con(cid:173)
`structed each containing selected BMA 031 amino
`acids within the human frameworks. The human(cid:173)
`ized genes were transfected into Sp2/0 hybridoma
`cells by electroporation and transfectomas secret (cid:173)
`ing humanized antibody were isolated. Levels of
`antibody expression up to 7 pg/cell/24 h were ob(cid:173)
`tained. The humanized antibody, BMA 031-EUCIV2,
`competed poorly with murine BMA 031 for binding
`to T cells. BMA 031-EUCIV3, however. bound specif(cid:173)
`ically t o T cells and competed effectively with both
`the murine BMA 031 antibody and a previously
`constructed chimeric BMA 031 antibody for binding
`to these cells. The relative affinity of BMA 031-
`EUCIV3 was about 2 .5 times lower than BMA 031.
`The ability to promote antibody dependent cell-me(cid:173)
`diated cytolys is was significantly enhanced with the
`engineered antibodies as compared t o murine BMA
`031. Huma nized BMA 031 is a clinically relevant,
`genetically engineered antibody with potential uses
`in transplantation. gra ft vs host disease, and auto(cid:173)
`immunity.
`
`mAb are emerging as a major modality for therapy of
`various pathologic conditions including malignant dis(cid:173)
`ease. cardiovascular disease, and autoimmune diseases.
`Some of these have demonstrated efficacy In treating
`colon carcinoma ( l ). B cell lymphomas (2), neu roblastoma
`(3). a nd in preventing transplant rejection (4. 5).
`Clinical trials with murine antibodies. although en(cid:173)
`couraging. have indicated at least two fundamental prob(cid:173)
`lems of antibody therapy. First, murlne IgG has a much
`shorter circulating half-life in man compared to what has
`been reported for human antibodies (6, 7), so that effec-
`
`Received for publication March 18. 1991.
`Accepted for publication September 16. 1991 .
`The costs of publicallon of this article were defrayed In part by the
`payment of page charges. This article must therefore be hereby marked
`aduerttsement ln accordance with 18 U.S.C. Section 1734 solely to Ind!·
`cate this fact.
`'Address correspondence and reprint requests to Or. C. W. Shearman.
`Sterling Drug. Inc .. 512 Elmwood Avenue. Sharon Hiii. PA 19079.
`•Present address Department of Molecular Genetics. Smith Kline Bee(cid:173)
`cham. 709 Swcdeland Road. King of Prussia. PA 19406.
`
`live mAb therapy may require frequent multiple treat(cid:173)
`ments with large amounts of murlne a ntibody. Second.
`administration of murlne IgG elicits a brisk HAMN1 re(cid:173)
`sponse that that can further reduce the circulating half(cid:173)
`life of the mAb and produce allergic reactions Including
`anaphylaxls (8-10).
`Almost all of the murin e mAb currently being used
`clinically provoke HAMA responses In patients. These
`Include HAMA against both the C region and the V region
`(11). HAMA responses lead to altered pharmacoklnetics
`of the Injected mAb. The antibody Is rapidly cleared from
`the serum and reduced antibody levels are attained ( 12).
`Although severe side effects are rare in patients with
`HAMA after retreatment with antibody, It Is clear that If
`mAb are to be used therapeutically, reliable methods
`must be devised to reduce Immune mediated complica (cid:173)
`tions or adverse reactions (13).
`One approach to better lmmunotheraples currently
`being explored is to produce a truly human antibody.
`Unfortunately, human mAb technology has lagged far
`beh ind that of murtne-based monoclonal technology. Hu(cid:173)
`man hybrldomas are difficult to prepare. are often un sta(cid:173)
`ble, and secrete antibody at low levels (14. 15). The mAb
`generated are usually of the IgM class and of low affi nity.
`An attractive and viable strategy Is to produce Mhuman(cid:173)
`ized· versions of murlne mAb through gen etic engineer(cid:173)
`ing. Methods have been devised to replace all regions of
`a murtne antibody with analogous human regions ( 16-
`18). Chimeric antibody technology has been applied to
`several thera peutically Important antibodies ( 19- 24) and
`has been useful In class switching and the production of
`isotypes with specific effector functions (25. 26). A chi(cid:173)
`meric a ntibody composed of the V regions of murlne mAb
`l 7 I A an d the human 'Y l C region has recently been used
`In patients with colon cancer. Whereas murine l 71A has
`been used extensively in clinical trials and elicits a very
`pronounced HAMA response that alters Its pharmacoki(cid:173)
`netlcs. antibody responses to chimeric l 7 1A have been
`dramatically reduced. Moreover. the circulating half-life
`was Increased relative to murine l 71A and higher serum
`levels could be maintained at lower Infused doses (27).
`Thus. with judicious genetic engineering. it Is possible to
`manipulate antibody pharmacoklnetlcs to minimize toxic
`side effects.
`Chimeric antibodies may be effective in lowering the
`HAMA response In patients and increasing serum ha lf(cid:173)
`lives. but these properties are sttll Inferior to human
`
`•Abbreviations used In this paper: HAMA. human anll-munnc anti(cid:173)
`body: ADCC. antibody dependent cell mediated cytolysis: FR. framework
`region.
`4366
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`1 of 8
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`Celltrion, Inc., Exhibit 1072
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`

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`HUMANIZED ANTl-T CELL ANTIBODIES
`
`4367
`
`antibodies. Inasmuch as chimeric antibodies are still 30%
`murlne, enhanced efficacy may be obtained by human(cid:173)
`izing the V regions. New technologies have recently been
`advanced to produce totally humanized antibodies by
`grafting the CDR of murine antibodies onto human FR
`(17, 28-30). The resulting antibodies when expressed
`with human C regions should be essentially human. This
`technology, although technically straightforward, Is not
`always totally successful. Selected FR amino acids ap(cid:173)
`pear to be Involved In Ag binding. Identification of Im(cid:173)
`portant FR amino acids has been achieved, up to now,
`by the use of x-ray crystallographic data ( 1 7) and sophis(cid:173)
`ticated computer modeling (30) and several totally hu(cid:173)
`manized antibodies have been produced with affinities
`close to those of their parental antibodies ( 1 7. 28-30).
`We report here the production of a humanized anti(cid:173)
`body. without the use of sophisticated structural data.
`which retains the affinity and specificity of BMA 031. a
`murlne mAb directed against the human a/(3 TCR. More(cid:173)
`over. humanized BMA 031 displays enhanced ADCC ac(cid:173)
`tivity. BMA 031 has been used successfully In preventing
`organ transpla nt rejection (5) and may have potential
`efficacy In other T cell-related disorders.
`
`MATERIALS AND METHODS
`
`Cell culture. The BMA 031 and Sp2/0-Agl4 hybridomas wel"e
`cultul"ed In DMEM media supplemented with l 0% FCS. 2 mM L(cid:173)
`glutamlne. 10 mM HEPES. pH 7.3. 10 mM nonessential amino acids
`(GlBCO. Gaithersburg. MD). and l 0 mM pyruvate. Chimeric and
`humanized BMA 031 transfectomas wel"e grown In the above media
`containing 1 µg/ml mycophenollc acid. 50 µg/ml xanthlne. and 500
`µgfml Genetlcln (GIBCO). All lines were maintained at 37°C In 7%
`C02.
`C-Omputer analysis. Sequences were manipulated and homology
`searches were performed with the Genetics Computer Group Se(cid:173)
`quence Analysis Software Package (University of Wisconsin Biotech(cid:173)
`nology Center. Madison. WI)) using the National Biomedical Research
`Foundation databases.
`Synthesis qf VH and VL regions. The VH and VL exons were
`synthesized on an Applied Biosystems (Foster City. CA) model 380A
`DNA synthesizer. Each region was synthesized completely as EcoRl(cid:173)
`HlndllJ fragments consisting of overlapping (10- 15 nucleotide over(cid:173)
`lap) ollgomers (75- 1 1 O nucleotides). The ollgomers were deprotected
`and purified by electroelutlon from polyacrylamlde gels. The ollgo(cid:173)
`mers were then mixed ln equlmolar amounts (30 pmol). phosphory(cid:173)
`lated. annealed, and ligated Into pUC 19 previously digested with
`EcoRI and Hlndlll.
`Nucleotide sequencing. DNA sequencing of the synthesized VH
`and VL regions was performed directly on pUC subclones using
`universal forward and reverse primers (31 ).
`Construction of humanized genes. To ensure efficient expres(cid:173)
`sion. the synthesized V regions were Inserted Into previously Isolated
`genomic fragments (24) In place of the murlne V regions. The re(cid:173)
`sulting 5.6-kb EcoRI VH fragment was cloned Into a mammalian
`expression vector containing the human 'Y 1 C region and the gpt
`gene for selection. The 3.0-kb HlndlU VL fragment was cloned Into
`a veclor containing the human KC region and the neo gene (see Fig.
`5).
`Transfectlon of DNA Into Sp2/0 cells by electroporatlon. DNA
`was Introduced Into murlne hybrtdoma Sp2/0-Agl 4 cells by electro(cid:173)
`poratlon. The I to 2 x 107 actively growing Sp2/0-Agl 4 cells were
`washed and resuspended In I .0 ml of sterile PBS. A total of I 5 µg of
`each humanized. lgK and lgGJ. plasmid (llnea.rlzed with BamHI) was
`added to the cell suspension. The DNA/ cells were transferred to a
`precooled shocking cuvette. Incubated on tee at least 5 min and then
`a 0 .5 kv/cm electric pulse was deUvered for 1 O ms (Transfector 300.
`STX. San Diego. CA). After shocking. the DNA/cell mixture was
`returned to Ice for 10 min and lhen diluted In 40 ml of supplemented
`DMEM and lncubaled at room temperature for 10 min. Finally. the
`cells were transferred to a 37°C lncubalor with 7% COz for 48 h
`before plating In selective medium. containing 1 µg/ml mycophenollc
`acid. 50 µg/ml xanthlne. and I mg/ml Genetlcln. Cells were plated
`In 96-well plates at 3 x Io• cells/well.
`Cytojluorometrlc assayjor affinity. To analyze the relative affin(cid:173)
`ities of murlne. chimeric. and humanized BMA 031 antibodies.
`
`competitive lmmunofluorescence assays were carried out. PBMC
`were separated by Flcoll-Hypaque density gradient centrifugation
`and incubated on Ice for I h In the dark with mAb at various
`concentrations (0.05-50 µg/ml) premixed with either FITC-BMA031
`or FITC-BMAEUCIV3 (2 µg/ml). Unbound antibodies were removed
`by two washing steps. Cells from all experiments were analyzed
`either on an Ortho (Raritan. NJ) Cytofluorograph 50H/2 I 50 Com(cid:173)
`puter System or on a Becton Dlck.lnson (Mountain View. CA) FACStar
`Plus as described elsewhere (32). The Intensity of fluorescence was
`calculated by modified Ortho or standard FACStar Plus software and
`Is expressed as mean channel number.
`Cytotoxicity assays. To measul"e the cr.;olytlc capacity of the
`BMA 031 antibody preparations. a 20 h I 'Crl release assay was
`performed to measure ADCC and NK activity. (51Crl-labeled HPB(cid:173)
`ALL target cells were Incubated with (AOCC) or without (NK activity)
`various concentrations of antibodies for 20 h In the presence of
`Flcoll-separated PBL (effector cells). a/{J TCR negative CEM cells
`were used as control target cells. The antibodies were allowed to
`bind first to target cells (30 min) before the effector cells were added.
`The E:T cell ratio varied from l:l to 50:1. Cytolysis In the absence
`of antibodies was considered to be due to NK activity. The percentage
`of specific lysls was calculaled as described earlier (33). Spontaneous
`15 'Cr) release In the absence of effectol" cells and In the presence of
`the antibodies being tested was always Jess than 53. All samples
`were analyzed In t ripllcalc.
`
`RESULTS
`
`Designing humanized BMA 031 antibodies. To deter(cid:173)
`mine the optimal human sequence with which to human-
`1.Ze the murlne BMA 031 antibody. the murlne BMA 031
`amino acid sequence was used to search the NBRF data
`base for the most homologous human antibody. Inas(cid:173)
`much as molecular models of antibodies show strong
`Interactions between the H and L chains, we decided to
`use the H and L chain from the same human antibody.
`The human EU antibody turned out to be the best overall
`choice. The homology between the BMA 031 and EU FR
`(nos. 1-3) was 79% (67% Identical) for the H chain and
`81 % (63% identical) for the L chain. The BMA 031 anti(cid:173)
`body uses JH3 and JK5. These are most homologous to
`human JH4 and JK4. A first generation humanized BMA
`031 antibody would contain BMA 031 CDR. EU FR. and
`homologous human J regions. We refer to this antibody
`as BMA 031-EUCIVl (Fig. 1).
`A refinement to this basic humanized version can be
`made in the sequence Immediately before and after the
`CDR. The CDR are assigned based on sequence homology
`data (34). Molecular models of antibodies have shown
`that the actual CDR loops can contain amino acids up to
`five amino acids away from the "Kabat" CDR (36). Also.
`Reichmann et al. (17) have shown the functional Impor(cid:173)
`tance of a FR amino acid four residues from a CDR.
`Therefore, maintaining at least the major amino acid
`differences (in size or charge) within four amino acids of
`the CDR as murlne may be beneficial. We refer to the
`antibody containing these changes as BMA 03 l -EUCIV2
`(Fig. 1 ). Additionally. all differences within four amino
`acids of the CDR could be maintained murlne. We refer
`to this antibody as BMA 031-EUCIV3.
`Further refinements can be made. but, without com(cid:173)
`plex computer modeling, It Is difficult to priorltlze their
`Importance. For example. several amino acids are either
`BMA 031 specific or EU specific (i.e., different from the
`consensus sequence within their subgroups). Inasmuch
`as these amino acids presumably arose through somatic
`mutation lo enhance their respective activities. it would
`seem logical to maintain the BMA 031-speclfic amino
`acids and change the EU-specific amino acids to the
`human consensus. But this can have potential adverse
`
`2 of 8
`
`Celltrion, Inc., Exhibit 1072
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`

`

`4368
`
`HUMANIZED ANTI-T CELL ANTIBODIES
`
`JO
`10
`50
`BU OVOLVQSGA!VKKPGSSVXVSCKASGGTPSRSAIIVVllQAPGOCLllVMGGIVPMPGPPNY
`BHA B Q P LV A M
`YK TSYVllB K K
`I Y N YNDVTK
`CIV-1
`SYVllB
`Y N YNDVTK
`CIV-2
`YK TSYVllB
`Y N YNDVTK
`C!V-3
`YK TSYVllB K
`I Y N YNDVTK
`\COii/
`\-----Cl>R-2
`1
`
`110
`90
`70
`BU AQK.POCRVTITADBSTNTAYMBLSSLRSBDTAPYPCAGG.YGIYSPBBY .. NGGLVTVSS
`BHA NB K KA L S K SS
`T S VBY R S YD DGPV VGQ T
`A
`CIV-1 NB K
`S YD DGPV VGO T
`CIV-2 NB K A
`R S YD DGl'V VGO T
`CtV-3 NB K KA L
`VBY R S YD DGFV VGO T
`- ----/
`\--CDR-3--/
`
`B
`
`50
`30
`10
`EU DIQMTOSPSTLSASVGDRVTITCRASQSINTVLAVYQQKPGKAPKLLHYKASSLESGVPS
`BHA O VL
`AIM
`P BK H S TS V. SYllB
`S TS RVI OT K A
`A
`CIV- 1
`S TS V.STHB
`OT K A
`CIV-2
`S TS Y.SYHB
`R OT KA
`CIV-3
`H S TS V.SYHB
`RVI OT K A
`\ --CDR-1--/
`\CDR-2/
`
`A
`A
`
`90
`70
`EU RPIGSGSGTBPTLTISSLQPDDPATYYCQOTIISDSKHPGOGTKVEVJC.
`BHA
`S
`SYS
`llEAB A
`VS NPLT A L L
`CIV-1
`VS NPLT G
`I
`CIV-2
`VS NPLT G
`I
`CIV-3
`VS NPLT G
`I
`\-CO!t-3-/
`
`Figure J. Amino acid sequences of EU. BMA 031, and humanized
`BMA 031 V regions. A. The VH region and B. the VL region. The positions
`of the CDR are Indicated.
`
`TABLE I
`Amino acid (AA) differences between BMA 031 and EU and their
`consensus sequences
`Human
`AA
`
`BMA031
`AA
`
`Mouse
`AA
`
`AA Position"
`
`EUAA
`
`H chaln. EU specific
`70
`lie
`Ala
`72
`74
`Glu
`93
`Phe
`Phe
`95
`98
`Gly
`
`L chain. EU specific
`Thr
`10
`48
`Met
`63
`lie
`70
`Glu
`81
`Asp
`
`H chain. BMA specific
`Gin
`I
`7
`Ser
`9
`Ala
`20
`Val
`40
`Ala
`Ala
`72
`82
`Glu
`94
`Tyr
`
`Val
`Tyr
`Arg
`
`Ser
`lie
`Ser
`Asp
`Glu
`
`Gin
`Ser
`Ala
`Val
`Ala
`•
`Glu
`Tyr
`
`Leu
`Ser
`Lys
`Val
`Tyr
`Arg
`
`lie
`Ile
`Ser
`Ser
`Glu
`
`Glu
`Ser
`Pro
`Met
`Lys
`Ser
`Glu
`His
`
`Leu
`Val
`Lys
`Val
`Tyr
`Arg
`
`lie
`Ile
`Ser
`Ser
`Glu
`
`Gin
`Pro
`Ala
`Leu
`Arg
`Val
`Gin
`Tyr
`
`L chain. BMA specific: None
`•Numbers correspond to those in Figure 1.
`•variable.
`
`consequences. Changing an amino acid in one chain may
`cause changes in the interactions with other amjno acids
`of that chain as well as with amino acids in the other
`chain. Therefore. extreme caution must be exercised to
`limit the number of changes. Table l outlines these po(cid:173)
`tential changes. The residue numbers correspond to
`those in Figure 1. As can be seen. EU differs from the
`human VH-1 subgroup consensus sequence in six posi(cid:173)
`tions. Three are within four amino acids of the CDR (nos.
`
`AA Position•
`
`H chain
`27
`28
`30
`38
`48
`67
`68
`70
`93
`94
`95
`98
`
`L chain
`21
`Met
`lie
`46
`Arg
`Leu
`47
`Trp
`Leu
`48
`lie
`Met
`Ala
`60
`Ser
`• Numbers correspond to those In Figure I .
`
`70. 95, and 98). and these are addressed in BMA-031-
`EUClV3. In one position (no. 93) the human consensus
`sequence is the same as BMA 031. Moreover, the Phe93
`in EU Is highly unusual; this amino acid Is only found in
`this position in one other human antibody in subgroup
`VH-m. One could rationalize changing this from EU to
`the human consensus. so we incorporated this change
`into BMA 031-EUCIV3. For the two remaining positions
`(nos. 72 and 74). there is no clear human consensus so
`we maintained the EU sequence. The L chain had five
`EU-specific amino acids. One is within four amino acids
`of the CDR (no. 48) and Is maintained as BMA 031 In
`BMA 031-EUCIV3. In two positions (nos. 63 and 81) the
`human consensus is the same as BMA 031 and therefore
`could be changed to the human consensus. We decided
`not to make these changes at this time. The other two
`positions (nos. l 0 and 70) were also not changed to limit
`the number of substitutions. There are eight BMA 031
`specific amino acids in the H chain. In two positions (nos.
`7 and 82) the BMA 031 sequence is the same as EU. His94
`is unique to BMA 031 . This position is considered Minvar(cid:173)
`iant" with Tyr9 4 occurring more than 98% of the time.
`Therefore. we decided to incorporate this change into
`BMA 03 l-EUCIV3. The remaining five positions (nos. 1.
`9. 20, 40. and 72) were maintained EU to limit the num(cid:173)
`ber of changes. There are no BMA 031 -speciftc amino
`acids in the L chain. The sequence is identical to the
`subgroup VI consensus. The changes in the human EU
`framework sequence back to BMA 031 are summarized
`in Table II. Twelve changes were made In the H chain; 5
`in BMA 031-EUCIV2 and 7 more in BMA 031-EUCIV3.
`Five changes were introduced into the L chain; two in
`BMA 031-EUCIV2 and three more in BMA 031-EUCIV3.
`Determination qf DNA sequence for humanized V
`regions. The amino acid sequence of the V regions were
`reverse translated using the actual BMA 031 codons
`wherever possible and BMA 031 codon preferences every(cid:173)
`where else. To aid In future modifications. unique restric(cid:173)
`tion enzyme sites were engineered into the sequence at
`approximately 60-bp intervals by making use of the de(cid:173)
`generacy of the genetic code. Finally. convenient restric(cid:173)
`tion enzyme sites 5' and 3' of the coding region of BMA
`031 were identified and this flanking sequence was In(cid:173)
`corporated into the final humanized sequence to be syn-
`
`TABLE II
`Amlno acid <AA) changes tn EU FR
`CIV2
`AA
`
`BMA03J
`AA
`
`EUAA
`
`Gly
`Thr
`Ser
`Arg
`Met
`Arg
`Val
`lie
`Phe
`Tyr
`Phe
`Gly
`
`Tyr
`Lys
`Thr
`Lys
`lie
`Lys
`Ala
`Leu
`Val
`His
`Tyr
`Arg
`
`CIV3
`AA
`
`Tyr
`Lys
`Thr
`Lys
`lie
`Lys
`Ala
`Leu
`Val
`His
`Tyr
`Arg
`
`Met
`Arg
`Trp
`lie
`Ala
`
`Tyr
`Lys
`Thr
`Arg
`Met
`Arg
`Ala
`lie
`Phe
`Tyr
`Phe
`Arg
`
`lie
`Arg
`Leu
`Met
`.Ala
`
`3 of 8
`
`Celltrion, Inc., Exhibit 1072
`
`

`

`HUMANIZED ANTl-T CELL ANTIBODIES
`
`4369
`
`theslzed. The final DNA sequences of BMA 031-EUCIV2
`VH and VL. excluding the EcoRI and Hlndlll cloning ends.
`are shown In Fig. 2.
`Synthesis of humanized BMA 031 V regions. The L
`and H chain V region exons encoding the humanized
`antibodies were synthesized completely as EcoRl-HindUI
`fragments consisting of 10 to 15 overlapping (10- 15
`nucleotide overlap) ollgomers (75- 110 nucleotides). The
`ollgomers were phosphorylated, annealed and ligated Into
`a pUC vector previously cut with EcoRI and Hlndlll. The
`assembled fragments were sequenced to verify accuracy
`of synthesis.
`Reconstruction of BMA 031 genomlcfragments with
`humanized V exons. To Increase the probability of effi(cid:173)
`cient expression of the synthesized coding regions. the
`humanized sequences were replaced Into the previously
`isolated 5.6-kb EcoRI VH and 3.0-kb Htndlll VL genomic
`fragments of BMA 031 (Fig. 3). Due to the lack of unique
`restriction enzyme sites, several subclonlngs were nec(cid:173)
`essary. To achieve this goal. four vectors. each containing
`modified genomic subfragments. were constructed. The
`first vector, pUCBMAVH-1.0HLiN was constructed by
`subclonlng the 1.0-kb HtndIII BMA 031 VH fragment Into
`pUC19 with subsequent deletion of the 5'-Nstl site. The
`second vector. pUCt.HBMAVH-5.6RLiH, was derived by
`cloning the 5 .6-kb EcoRI BMA 031 VH fragment Into a
`pUC19 vector with a previously deleted HindIII site. The
`5' -HtndIII site of the insert was then deleted to complete
`the construction. The third vector, pUCBMAVL-l.4RH2.
`was constructed by subclonlng the 1.4-kb EcoRI-Hlncll
`BMA 031 VL fragment Into pUC19. The fourth vector,
`pUCLiRSBMAVL-3.0H. was made by cloning the 3.0-kb
`Hlndlll BMA 031 VL fragment Into a pUC19 vector that
`had a previous deletion from the EcoRI site to the Sall
`site In the polylinker.
`The cloning scheme to replace the humanized se(cid:173)
`quences Into the genomic fragments Is outlined in Figure
`4. The newly synthes ized Saul-Nsfl BMA 0 3 1-EUCIV2
`
`VH fragment was isolated from the pUCl 9 subclone and
`cloned into pUCBMAVH-1.0HLiN. Then. the 1.0-kb
`HindllI fragment was isolated and cloned into
`pUCLiHBMAVH-5.6RLiH. Finally, the 5.6-kb EcoRI frag(cid:173)
`ment was isolated and subcloned Into the mammalian
`expression vector containing the human 'Y 1 C region and
`the gpt gene for selection (Fig. 5).
`The newly synthesized Saul-Htncn BMA 03 I -EUCIV2
`VL fragment was Isolated and cloned Into pUCBMA VL-
`1 .4RH2. Then. the 1.4-kb EcoRI-HtncII fragment was
`Isolated and cloned Into pUCLiRSBMAVL-3.0H. Finally.
`the 3.0 HlndIII fragment was Isolated and cloned Into the
`mammalian expression vector containing the human K C
`region and the neo gene for selection (Fig. 5).
`The BMA 03 l -EUCIV3 constructs were prepared in the
`same manner as BMA 031 -EUCIV2. Replacement ollgo(cid:173)
`mers Incorporating the coding changes for BMA 031-
`EUCIV3 were synthesized and cloned Into the pUCBMA(cid:173)
`EUClV2 constructs. The final clone was sequenced to
`ensure accuracy of the coding sequence. The BMA 031-
`EUCIV3 V regions were replaced Into the original BMA
`0 3 1 genomic fragments and these fragments were cloned
`Into the mammalian expression vectors described above.
`Expression and purification of humanized BMA 031
`antibodies. The humanized genes were transfected Into
`Sp2/ 0 hybrtdoma cells by electroporatlon and selected In
`media containing both mycophenollc acid and Genetlcln.
`Transf ectomas secreting humanized BMA 031 antibodies
`were identified by ELISA. Secretion levels up to 7 pg/cell/
`24 h were obtained. The best clone from each transfec(cid:173)
`tion (CIV2 and CIV3), with respect to secretion level and
`growth characteristics, was expanded for further study.
`The BMA 031 -EUCIV2 and -EUCIV3 antibodies were
`partially purified by protein A-Sepharose column chro(cid:173)
`matography. Analysis of the antibodies by reducing and
`nonreducing SDS-PAGE showed a high degree of purity
`(data not shown). Analysis by a series of ELISA assays
`showed that the antibodies contained human K and 'Y 1 C
`
`... '
`~AC'TGTAGA~~~ • • •
`
`.,.. t
`
`1
`
`I
`~TCACAtt'T~TTCTCt~C'1'C.AGC'TCC
`l -----.. · - - - - - - - - - -.. ·-------- -·-·--·------· 60
`
`l
`
`S...c.I
`I
`I
`&ACTAG'TTCACT~~t"f'CtGAC~
`,20
`•• ------·· · ··· ······· ·····--·· ··--·-·· · - -······-· -·-····--
`r.cA~~~et~
`L\tO S ' A
`l VK K P C:SS WJ(, V S C k
`ltpM 11
`Ora UI
`I
`I
`AQQCftCCQ;ATATMA nCACTAGCfA TCTTATGCM:'fGOCTC~CCTtcCC
`................ _ _____ .. .................. ................ .
`................ _____
`~ATATTTAAC'TC.ATCIC4t lll:.MTA~~ACCGC
`A. SC TK PTS TV M
`II V 'fA Q A.P GO
`
`S.u I
`
`Ace I
`
`l'Co l
`I
`I
`I
`CCTCAOCTMCAGAGCGCAC , •. . .. . 140 ~p • . ... . . C'l'TT'TTTeCnTtACf
`··-----···--·-·---· ------- ·------ ---· ---------·· - - - -·
`CCACTCCAmtCTQOCCTC . .... ,, •••• • ,., , • • ••• c:•4°•.-ccu 0
`•4r•TCA
`l coa v
`
`llO
`
`I
`TCAGCAC~TCTATCAGACATCCAAACrGIXTTC
`l60
`.................. ....... _
`........... __ ___ • ___________ _ . _ _______ ._
`
`I
`CATMTA~TATCCACA1'CACCCAATCTCCATOC.liCCC'tC.&CTCCA.TCTCT
`·-·-···- · -·-·--· ·-------.. - --- - --· ---------· -------· 240
`CTA1TATAGGTCTCCTCTATACCTCT4C'J'CICc;TT4CAQCTACCICCC~~
`D1 0
`Jt TOS t
`S T LS A S Y
`Tfhl lt I
`I
`~eACCATCA!~MCTTl.Ut"OCACro;u
`24l --------- ----·· ·--------~-·----·····--------· JOO
`~A.CT«.Ac:r;r~ncu.lC'l'~T
`C P ~ V T K T C S A T S S V S 1
`l
`I V T
`A• a I
`\--------cta-1----···-/
`
`181
`
`JOl
`
`121
`
`180
`
`Do 1
`
`'- -coa-•---1
`
`Figure 2. DNA sequences of the V r e(cid:173)
`gions of BMA 0 3 1-EUCIV2. A . The BMA
`031-EUCIV2 VH region and B. the BMA
`031-EUCIV2 VL region.
`
`111
`
`241
`
`)00
`
`I
`~J"CG.C.ATATAnAAn:atACM'fCATc:TTACTMJ:rACMTC<ACA
`..... ......... . ____ .____ 140
`............... ................ . ........ ..... . ...................
`TC~ACCC'TATATU.nAOCMTCnAC'TACMTCAntATcn li&TCf
`CLl Vlll CTl ffP Y
`M O VT~ T M
`l l
`\ ....................... ceot.. 2 ............ ___ ... .
`XH IH
`I
`~ACMnAc:cc;cclCACC.ACTCCAC!'MfACACCCTACJ.TOCMCC
`.............. . __ ,. __ ...................... .................
`.............. . .................
`tc:.ucmca:tcccccn:nAA~·,,.·TCTCXiCATCTAccrcc
`r-. c:a 1.Tt T AO IS 'Tlf t A 't' JC& L
`111 It
`________ ,
`
`I
`~TCTCAOCACAC'TGOCTTc:'T A~Co\OOC.AOt'Tl+CTA1'C
`:JO) - ------· ·· - ····· · · ····--·--· ---------·
`................ ..................
`ACTCCTC.GC4CTC'l'~CMCAT~4CACCM'CTCCC'T<lCATCAT4C
`SSL lS I O TAPT , C AJ C:SfT O
`(pn 1
`\------c:Dl·l·
`
`AttAOl:AC«.C'n'TCTn~ACCCT~AGCTCACT
`161 --···--·· ·• ·-- ·····-· ---------· -······· · ···-···· '"'·-·····-·
`TMTOCT~~~C:.C~C:CACTCA
`Y OCPYfVC: OC TLV TYSS
`··-·----....... ..-.. .... /
`~·•t I
`I
`CCTAACTTCTCOCAncTAM~TCTT
`
`'
`
`421
`
`411
`
`360
`
`410
`
`~ACA.UCTCTC'f~C:CC.U.C
`0 0
`l
`, C I A P l
`l L M T D T 5
`l L 4 S
`\ -----cm.-2 ....... /
`•••• u
`
`I
`TCCACTCCCTCOCOCC'TtCA~
`)tl --·······~-----... · - - - - -· - - - -- --
`.,.,
`ACCTCAQQCACGCCCCAACT~ACTC
`CVP A lP lC : s c ; s c ;11 r TL t lS
`
`.... ,
`
`I
`CACCCTCCACICCACATCAnTCOC't'•a:n.n~u.ccc:cct
`............ - .... ·-···--- · · - - - ·· - - - -------- 4IO
`CTCQGA~ACT~TGCAtM~TTCCO:C.A
`SL.QPOO PA TT lCOO V S SWP L
`........ - cca .. >--- ...
`lJ1ft i
`\ .. -
`I
`CA~~n~TCTmT
`................................... -
`····· -··-- - -----·--···· · ··- - - )40
`~CC.A~...- rTTOCA~llCAAAAA
`T P CCCTCY &
`I ~
`•I
`
`ft4tCTC'tMC~tcn~~~
`· ··-·-·· · ..... - · · · · · · · - - -· - - · · - - - - 600
`)4t - - - - · · ·· ·········· -
`U.lAC.c:.t.~ACAATCCT~ACMCTCT
`ltac U
`
`I
`MA~nAteAcTrc.t.c
`- -··-··•----·- •·- ··-- Ut
`TTT~MtAC'TCMCTC
`
`IOI
`
`4 of 8
`
`Celltrion, Inc., Exhibit 1072
`
`

`

`4370
`
`HUMANIZED ANTl-T CELL ANTIBODIES
`
`A
`
`R
`
`II
`
`8
`
`llff
`
`s
`
`N 8 SS
`
`p R
`
`1. 0 kb
`
`8
`
`R
`
`s p
`
`82 p
`
`SN H
`
`1.0 kb
`
`Figure 3. Partial restriction enzyme maps of BMA 031 V regions. A.
`The 5 .6 -kb EcoRI VH fragm ent containing the vou. exon. B. the 3.0 ·kb
`H!ndlll VL fragment contaJnlng the VJ• exons. H. Hlndlll: H2. Hlncll: N.
`Nsll; P. Pstl; R. EcoRI: S. Saul.
`
`regions. Moreover, the antibodies did not react with anti(cid:173)
`murlne antibodies (data not shown).
`Characterization of humanized BMA 031 antibodies.
`The BMA 031-EUCIV2 antibody bound poorly to T cells.
`ln contrast. BMA 031-EUCIV3 shows an Identical speci(cid:173)
`ficity as murlne BMA 031 . They both bind specifically to
`T cells and show no reactivity toward monocytes. E. or
`granulocytes (data not shown).
`The relative affinities of murtne BMA 031. chimeric
`BMA 031 (human lgGl). and the humanized variants
`were compared by competitive tmmunofluorescence as-
`
`A
`
`says. The data shown In Figure 6 indicate that both the
`murine BMA 031 antibody and the previously con(cid:173)
`structed chimeric BMA 031-Gl antibody block the bind(cid:173)
`ing of BMA 031-FITC In the same dose-dependent man(cid:173)
`ner. BMA 031-EUCIV3 was about 2.5 times less efficient
`than murtne BMA 031. BMA 031-EUCIV2 was unable to
`totally block BMA 031-FITC binding, even at concentra(cid:173)
`tions as high as 50 µg/ml.
`BMA 031 has been shown to be poor at mediating ADCC
`using human effector cells. To evaluate the ADCC capac(cid:173)
`ity of the humanlz.ed antibodies. we compared them to
`rabbit anti-GH-1 antiserum. This antiserum was the best
`of eight rabbit anti-human T cell globulins In ADCC
`capacity. As shown In Figure 7. both the chlmerlc BMA
`031 antibody and the BMA 031-EUCIV3 antibody were
`very efficient at ADCC. Even at very low effector:target
`cell ratios (Fig. 7 A) or extremely low antibody concentra(cid:173)
`tions (Fig. 7. B and C). the engineered antibodies are
`highly potent at mediating killing of the HPB-ALL cells.
`
`DISCUSSION
`We have joined the DNA segments containing the CDR
`from the BMA 031 mAb specific for the a/{JTCR and the
`FR from the human EU antibody to the DNA segments
`encoding human -y-1 and " C regions. When the human(cid:173)
`ized genes were introduced Into non-lg producing Sp2/0
`cells, functional humanized antibodies specific forTcells
`were assembled and secreted.
`Functional antibody. however. was dependent on sub(cid:173)
`stitution of various murlne FR amino acids into the hu(cid:173)
`man FR. The Identification of important FR amino acids
`in the abs ence of structural data or computer models is
`difficult but. by careful analysis of antibody sequence
`homologies, it is possible to generate a humanized se(cid:173)
`quence with a high probability of maintaining Ag bind(cid:173)
`ing. Our method consists of three parts. First, and pos(cid:173)
`sibly most important, is starting with the human anti(cid:173)
`body most homologous to the murine antibody under
`
`Ftgure 4. The cloning scheme to re·
`generate the BMA 031 genomic fragments
`with the humanized V regions. A. Substi(cid:173)
`tuting the humanized VH region Into the
`5 .6-kb EcoRI VH fragment. BH. BMA 031
`VH exon: CH. humanized BMA 031 VH
`exon: pl. pUCBMAVH-1.0HAN: p2.
`p3.
`pUCBMACIVH:
`pUCBMACIVH·
`LOHAN: p4. pUCAHBMAVH-5.6RMI: p5.
`pUCAHBMACIVH-5 .6RAH: p6. pSV2gpt·
`hu')' I; p7, pSV2gpt-BMACIVH-hu')'I. B.
`Substituting the humanized VL region
`Into the 3 .0 HLndlll VL fragm ent. BL. BMA
`031 VL exon; CL. humanized BMA 031
`VL exon: p8. pUCBMAVL- l.4RH2: p9.
`pUCBMACIVL:
`plO.
`pUCBMACIVL(cid:173)
`l .4RH2: pl J. pUCARSBMAVl.,-3.0H: p 12.
`pUCARSBMACIVL-3.0H: pl3. pSV2neo(cid:173)
`hux; pl4. pSV2neo-BMACIVL-huK. Re(cid:173)
`striction enzyme sites Identified are: H,
`Hlndlll; H2. H!ncll: N. Nsll: R. EcoRI: s.
`Saul.
`
`~ Cl
`~~:.;:/ __ :
`
`_,,.,.
`
`GI.
`
`~~ ~
`(:) fCT,;:;,,/-0
`
`bolot•
`CL
`
`holot•
`".etof
`
`~
`8 -- (:)
`''°'°'·"' ..,.. /"""".
`....ctOI ~ /-cL
`"
`
`l
`
`•
`
`5 of 8
`
`Celltrion, Inc., Exhibit 1072
`
`

`

`HUMANIZED ANTl-T CELL ANTIBODIES
`
`4371
`
`100
`
`A
`
`R
`
`B
`
`R
`
`BllA 031-&UCIVB
`
`BllKAN CAl!Klo I
`
`p5V2- gpt
`
`80
`
`...J
`
`60
`
`• .. >-
`c • 40
`e • A.
`
`--0-- antl·GH 1
`BMA031
`SMA031-G1
`- -0 - BMA·EUCIV3
`-6 - - NK-aetlYity
`
`20
`
`0
`
`0
`
`10
`
`20
`
`30
`
`40
`
`50
`
`E:T
`
`Retlo
`
`H
`
`8
`
`H
`
`e
`
`A
`
`R
`
`B
`
`H
`
`BKA 031-BUCIVL
`
`BllKAN KAPPA
`
`pSV2- neo
`
`Ftgure5. Ei<presslon vectors for humanized BMA 031 V regions. A.
`The H chain expression vector containing the humanized BMA 03 1 VH
`regton. the human "Y I C region. and the guanine phosphoribosyl trans(cid:173)
`ferase gene for selection. B. The L chain expression vector containing the
`humanized BMA 031 VL region. the human• C region and the neomycin
`resistance gene for selection .
`
`100
`
`80
`
`• .. >-. j 60
`c • e •o
`• A.
`
`20
`
`--0--
`
`100ng/ml
`
`---- 10 ng/ml
`
`--.--
`
`- -0 - 1 ng/ml
`0.1 ngfml
`-6