0022-1767/91/14712-4366802.00/0
`THE JOURNAL OF IMMUNOLOGY
`Copyright © 1991 by The American Association of 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 a/6 T CELL RECEPTOR
`
`CLYDE W. SHEARMAN,'* DAN POLLOCK,* GARY WHITE,* KATHY HEHIR,*
`GORDON P. MOORE,”* E. J. KANZY,'anp ROLAND KURRLE'
`
`From the *Genzyme Corporation, Framingham, MA 01701; and *Behringwerke Aktiengeselischaft, D-3550
`Marburg, West Germany
`
`Completely humanized antibodies with specificity
`for the human a/§ TCR have been produced by ge-
`netic engineering. The L and H chain V region exons
`encoding the murine mAb BMA 031 CD regions and
`human EU framework regions were synthesized and
`replaced into previously isolated genomic frag-
`ments. These fragments were inserted into mam-
`malian expression vectors containing the human x
`and y 1 C region exons. Two variants were con-
`structed each containing selected BMA 031 amino
`acids within the human frameworks. The human-
`ized genes were transfected into Sp2/0 hybridoma
`cells by electroporation and transfectomas secret-
`ing humanized antibody were isolated. Levels of
`antibody expression up to 7 pg/cell/24 h were ob-
`tained. The humanized antibody, BMA 031-EUCIV2,
`competed poorly with murine BMA 031 for binding
`to T cells. BMA 031-EUCIV3, however, bound specif-
`ically to 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-
`diated cytolysis wassignificantly enhanced with the
`engineered antibodies as compared to murine BMA
`031. Humanized BMA 031 is a clinically relevant,
`genetically engineered antibody with potential uses
`in transplantation, graft vs host disease, and auto-
`immunity.
`
`tive mAb therapy may require frequent multiple treat-
`ments with large amounts of murine antibody. Second,
`administration of murine IgG elicits a brisk HAMA? re-
`sponse that that can further reducethecirculating half-
`life of the mAb and produceallergic reactions including
`anaphylaxis (8-10).
`Almost all of the murine mAb currently being used
`clinically provoke HAMAresponses in patients. These
`include HAMAagainst both the C region andthe V region
`(11). HAMAresponseslead to altered pharmacokinetics
`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
`HAMAafter retreatment with antibody,it is clear thatif
`mAb are to be used therapeutically, reliable methods
`must be devised to reduce immune mediated complica-
`tions or adverse reactions (13).
`One approach to better immunotherapies currently
`being explored is to produce a truly human antibody.
`Unfortunately, human mAb technology has lagged far
`behind that of murine-based monoclonal technology. Hu-
`man hybridomasare difficult to prepare, are often unsta-
`ble, and secrete antibody at low levels (14, 15). The mAb
`generated are usually of the IgM class andof low affinity.
`An attractive and viable strategy is to produce “human-
`ized” versions of murine mAb through genetic engineer-
`ing. Methods have been devised to replace all regions of
`a murine antibody with analogous humanregions (16-
`18). Chimeric antibody technology has been applied to
`several therapeutically important antibodies (19-24) and
`has been useful in class switching and the production of
`isotypes with specific effector functions (25, 26). A chi-
`meric antibody composed of the V regions of murine mAb
`171A and the human 7 1C region has recently been used
`in patients with colon cancer. Whereas murine 171A has
`been used extensively in clinical trials and elicits a very
`pronounced HAMAresponsethatalters its pharmacoki-
`netics, antibody responses to chimeric 171A have been
`dramatically reduced. Moreover, the circulating half-life
`wasincreased relative to murine 171A and higher serum
`levels could be maintained at lower infused doses (27).
`Thus, with judicious genetic engineering, it is possible to
`manipulate antibody pharmacokinetics to minimize toxic
`side effects.
`Chimeric antibodies may be effective in lowering the
`HAMAresponsein patients and increasing serum half-
`lives, but these properties are still inferior to human
`3 Abbreviations used in this paper: HAMA, human anti-murineanti-
`body; ADCC, antibody dependentcell mediated cytolysis; FR, framework
`region.
` Pfizer v. Genentech
`Pfizer v. Genentech
`4366
`IPR2017-01489
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`Genentech Exhibit 2033
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`
`mAb are emerging as a major modality for therapy of
`various pathologic conditions including malignant dis-
`ease, cardiovascular disease, and autoimmunediseases.
`Some of these have demonstrated efficacy in treating
`colon carcinoma(1), B cell lymphomas(2), neuroblastoma
`(3), and in preventing transplantrejection (4,5).
`Clinical trials with murine antibodies, although en-
`couraging, have indicated at least two fundamental prob-
`lems of antibody therapy. First, murine IgG has a much
`shortercirculating 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 publication of this article were defrayed in part by the
`paymentof page charges. This article must therefore be hereby marked
`advertisementin accordance with 18 U.S.C. Section 1734 solely to indi-
`cate this fact.
`} Address correspondence andreprint requests to Dr. C. W. Shearman,
`Sterling Drug, Inc., 512 Elmwood Avenue, Sharon Hill, PA 19079.
`? Present address Department of Molecular Genetics, SmithKline Bee-
`cham, 709 Swedeland Road, King of Prussia, PA 19406.
`
`

`

`HUMANIZED ANTI-T CELL ANTIBODIES
`
`4367
`
`antibodies. Inasmuchaschimeric antibodiesarestill 30%
`murine, enhanced efficacy may be obtained by human-
`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 humanC regions should be essentially human. This
`technology, although technically straightforward, is not
`alwaystotally successful. Selected FR amino acids ap-
`pear to be involved in Ag binding. Identification of im-
`portant FR amino acids has been achieved, up to now,
`by the use of x-ray crystallographic data (17) and sophis-
`ticated computer modeling (30) and several totally hu-
`manized antibodies have been produced with affinities
`close to those of their parental antibodies (17, 28-30).
`Wereport here the production of a humanized anti-
`body, without the use of sophisticated structural data,
`which retains theaffinity and specificity of BMA 031, a
`murine mAbdirected against the human a/8 TCR. More-
`over, humanized BMA 031 displays enhanced ADCC ac-
`tivity.BMA 031 has been used successfully in preventing
`organ transplant rejection (5) and may have potential
`efficacy in other T cell-related disorders.
`
`MATERIALS AND METHODS
`
`Cell culture. The BMA 031 and Sp2/0-Agl14 hybridomas were
`cultured in DMEM media supplemented with 10% FCS, 2 mM L-
`glutamine, 10 mM HEPES, pH 7.3, 10 mM nonessential aminoacids
`(GIBCO, Gaithersburg, MD), and 10 mM pyruvate. Chimeric and
`humanized BMA 031 transfectomas were grown in the above media
`containing 1 »g/ml mycophenolic acid, 50 ug/ml xanthine, and 500
`ug/ml Geneticin (GIBCO). All lines were maintained at 37°C in 7%
`CO..
`Computer analysis. Sequences were manipulated and homology
`searches were performed with the Genetics Computer Group Se-
`quence Analysis Software Package (University of Wisconsin Biotech-
`nology Center, Madison, WI)} using the National Biomedical Research
`Foundation databases.
`Synthesis of VH and VL regions. The VH and VL exons were
`synthesized on an Applied Biosystems(Foster City, CA) model 380A
`DNAsynthesizer. Each region was synthesized completely as EcoRI-
`HindiIll fragments consisting of overlapping (10-15 nucleotide over-
`lap) oligomers (75-110 nucleotides). The oligomers were deprotected
`and purified by electroelution from polyacrylamide gels. The oligo-
`mers were then mixed in equimolar amounts (30 pmol), phosphory-
`lated, annealed, and ligated into pUC 19 previously digested with
`EcoRI and Hindiil.
`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-
`sion, the synthesized Vregions were inserted into previously isolated
`genomic fragments (24) in place of the murine V regions. The re-
`sulting 5.6-kb EcoRI VH fragment was cloned into a mammalian
`expression vector containing the human y1 C region and the gpt
`genefor selection. The 3.0-kb Hindlll VL fragment was cloned into
`a vector containing the human « C region and the neo gene(see Fig.
`5).
`
`Transfection of DNA into Sp2/0 cells by electroporation. DNA
`wasintroduced into murine hybridoma Sp2/0-Ag14 cells by electro-
`poration. The 1 to 2 x 10” actively growing Sp2/0-Ag14 cells were
`washed and resuspendedin 1.0 ml of sterile PBS. A total of 15 ug of
`each humanized, Igx and IgG1, plasmid (linearized with BamHI) was
`added to the cell suspension. The DNA/cells were transferred to a
`precooled shocking cuvette, incubated on ice at least 5 min and then
`a 0.5 kv/em electric pulse was delivered for 10 ms (Transfector 300,
`BTX, San Diego, CA). After shocking, the DNA/cell mixture was
`returnedto ice for 10 min and thendiluted in 40 ml of supplemented
`DMEMandincubated at room temperature for 10 min. Finally, the
`cells were transferred to a 37°C incubator with 7% CO, for 48 h
`before plating in selective medium, containing 1 ug/ml mycophenolic
`acid, 50 ug/ml xanthine, and 1 mg/ml Geneticin. Cells were plated
`in 96-well plates at 3 x 10* cells/well.
`Cytofluorometric assayfor affinity. To analyze therelative affin-
`ities of murine, chimeric, and humanized BMA 031 antibodies,
`
`competitive immunofluorescence assays were carried out. PBMC
`were separated by Ficoll-Hypaque density gradient centrifugation
`and incubated on ice for 1 h in the dark with mAb at various
`concentrations (0.05—50 ug/ml) premixed with either FITC-BMA031
`or FITC-BMAEUCIVS (2 yg/ml). Unbound antibodies were removed
`by two washing steps. Cells from all experiments were analyzed
`either on an Ortho (Raritan, NJ) Cytofluorograph 50H/2150 Com-
`puter System or on a Becton Dickinson (Mountain View, CA) FACStar
`Plus as described elsewhere (32). The intensity of fluorescence was
`calculated by modified Ortho or standard FACStarPlus software and
`is expressed as mean channel number.
`Cytotoxicity assays. To measure the cytolytic capacity of the
`BMA 031 antibody preparations, a 20 h [*'Cr] release assay was
`performed to measure ADCC and NKactivity. [°'Crj-labeled HPB-
`ALLtarget cells were incubated with (ADCC) or without (NK activity)
`various concentrations of antibodies for 20 h in the presence of
`Ficoll-separated PBL (effector cells). a/8 TCR negative CEM cells
`were used as control target cells. The antibodies were allowed to
`bindfirst to target cells (30 min) before the effector cells were added.
`The E:T cell ratio varied from 1:1 to 50:1. Cytolysis in the absence
`of antibodies was considered to be due to NK activity. The percentage
`of specific lysis was calculated as describedearlier (33). Spontaneous
`{©'Cr] release in the absenceof effector cells and in the presence of
`the antibodies being tested was always less than 5%. All samples
`were analyzedin triplicate.
`
`RESULTS
`
`Designing humanized BMA 031 antibodies. To deter-
`minethe optimal human sequence with which to human-
`ize the murine BMA 031 antibody, the murine BMA 031
`amino acid sequence was used to search the NBRF data
`base for the most homologous human antibody. Inas-
`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 EUantibody 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-
`body uses JH3 and JK5. These are most homologousto
`human JH4 and JK4. A first generation humanized BMA
`031 antibody would contain BMA 031 CDR, EU FR, and
`homologous humanJ regions. Werefer to this antibody
`as BMA 031-EUCIV1 (Fig. 1).
`A refinement to this basic humanized version can be
`made in the sequence immediately before and after the
`CDR. The CDRare 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,
`Reichmannet al. (17) have shown the functional impor-
`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 murine may be beneficial. We refer to the
`antibody containing these changes as BMA 031-EUCIV2
`(Fig. 1). Additionally, all differences within four amino
`acids of the CDR could be maintained murine. Werefer
`to this antibody as BMA 031-EUCIV3.
`Further refinements can be made, but, without com-
`plex computer modeling, it is difficult to prioritize 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 to enhancetheir respective activities, it would
`seem logical to maintain the BMA 031-specific amino
`acids and change the EU-specific amino acids to the
`human consensus. But this can have potential adverse
`
`

`

`4368
`
`HUMANIZED ANTI-T CELL ANTIBODIES
`
`50
`30
`10
`EU QVQLVQSGAEVKKPGSSVKVSCKASGGTFSRSAIIWVROAPGOGLEWMGGIVPMFGPPNY
`BMA
`E
`Q PL A
`4M
`YK TSYVMH
`K K
`I Y N YNDVTK
`CIV-1
`SYVMH
`Y N YNDVIK
`cIv-2
`YK TSYVMH
`Y N YNDVTK
`cIV-3
`YK TSYVMH
`IY N YNDVTK
`\CDR/
`\-----CDR-2
`1
`
`110
`90
`70
`EU AQKFQGRVTITADESTNTAYMELSSLRSEDTAFYFCAGG. YGIYSPEEY. .NGGLVTVSS
`A
`BMA NE K KAL SK SS
`T
`S$ VHY RS YD DGFV Wed T
`CIV-1 NE K
`S YD DGFV WGQ T
`CIV-2
`NE K A
`RS YD DGFV WGQ T
`CIV-3
`NE KKAL
`VEY R S YD DGFV Wad T
`-----/
`\--CDR-3--/
`
`K
`
`B
`
`
`
`50
`30
`EU DIQMTQSPSTLSASVGDRVTITCRASQSINTWLAWYQQKPGKAPKLLMYKASSLESGVPS
`BMA
`Q VL
`AIM
`PEK M
`S TS V.SYMH
`STS RWI DITKA
`A
`CIv-1
`S TS V.SYMH
`DI KA
`CcIVv-2
`S TS V.SYMH
`ODIKA
`R
`CIV-3
`oS TS V.SYMH
`RWI DITKA
`\--CDR-1--/
`\CDR-2/
`
`A
`A
`
`M
`
`90
`79
`EU RFIGSGSGTEFTLTISSLOPDDFATYYCQQYNSDSKMFGQGTKVEVK
`BMA
`s
`SYS
`MEAE A
`WS NPLT
`A
`LL
`CIV-1
`WS NPLT
`G
`I
`CIV-2
`WS NPLT
`G
`I
`CIV-3
`WS NPLT
`G
`I
`\-CDR-3-/
`
`Figure 1. Amino acid sequences of EU, BMA 031, and humanized
`BMA 031 V regions. A, The VHregion and B, the VL region. The positions
`of the CDRare indicated.
`
`TABLE I
`Amino acid (AA) differences between BMA 031 and EU and their
`consensus sequences
`Human
`AA
`
`a
`
`EU AA
`AAPosition’
`H chain, EU specific
`70
`lle
`72
`Ala
`74
`Glu
`93
`Phe
`95
`Phe
`98
`Gly
`
`L chain, EU specific
`10
`Thr
`48
`Met
`63
`Ile
`70
`Glu
`81
`Asp
`
`BMA031
`AA
`
`Mouse
`AA
`
`>
`P
`P
`Val
`Tyr
`Arg
`
`Ser
`Ne
`Ser
`Asp
`Glu
`
`Leu
`Ser
`Lys
`Val
`Tyr
`Arg
`
`Tle
`Ne
`Ser
`Ser
`Glu
`
`Leu
`Val
`Lys
`Val
`Tyr
`Arg
`
`Ne
`lle
`Ser
`Ser
`Glu
`
`H chain, BMA specific
`1
`Gln
`7
`Ser
`9
`Ala
`20
`Val
`40
`Ala
`72
`Ala
`82
`Glu
`94
`Tyr
`
`Gln
`Glu
`Gin
`Pro
`Ser
`Ser
`Ala
`Pro
`Ala
`Leu
`Met
`Val
`Arg
`Lys
`Ala
`Val
`Ser
`®
`Gin
`Glu
`Glu
`Tyr
`His
`Tyr
`
`L chain, BMA specific: None
`* Numberscorrespondto those in Figure 1.
`> Variable.
`
`consequences. Changing an amino acid in one chain may
`cause changesin the interactions with other amino 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 I outlines these po-
`tential changes. The residue numbers correspond to
`those in Figure 1. As can be seen, EU differs from the
`human VH-I subgroup consensus sequence in six posi-
`tions. Three are within four aminoacidsof the CDR (nos.
`
`70, 95, and 98), and these are addressed in BMA-031-
`EUCIV3. In one position (no. 93) the human consensus
`sequence is the same as BMA 031. Moreover, the Pheg3
`in EU is highly unusual; this amino acid is only found in
`this position in one other human antibody in subgroup
`VH-III. 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 consensusis 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. 10 and 70) were also not changedto limit
`the numberof substitutions. There are eight BMA 031
`specific amino acids in the H chain. In twopositions (nos.
`7 and 82) the BMA 031 sequenceis the same as EU. HisSo4
`is unique to BMA 031. This position is considered “invar-
`iant” with Tyrg, occurring more than 98% of the time.
`Therefore, we decided to incorporate this change into
`BMA 031-EUCIV3. The remaining five positions (nos. I,
`9, 20, 40, and 72) were maintained EU to limit the num-
`ber of changes. There are no BMA 031-specific 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 Il. 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 of DNA sequence for humanized V
`regions. The amino acid sequence of the V regions were
`reverse translated using the actual BMA 031 codons
`whereverpossible and BMA 031 codon preferencesevery-
`whereelse. To aid in future modifications, unique restric-
`tion enzyme sites were engineered into the sequence at
`approximately 60-bp intervals by making use of the de-
`generacy of the genetic code. Finally, convenient restric-
`tion enzymesites 5’ and 3’ of the coding region of BMA
`031 were identified and this flanking sequence wasin-
`corporated into the final humanized sequenceto be syn-
`TABLE II
`
`Amino acid (AA) changes in EU FR
`a
`BMAO31
`cIv2
`Civ3
`
`AAPosition’
`EU AA
`AA
`nw
`AR
`H chain
`27
`28
`30
`38
`48
`67
`68
`70
`93
`94
`95
`98
`L chain
`Met
`He
`Met
`lle
`21
`Arg
`Arg
`Arg
`Leu
`46
`Trp
`Leu
`Trp
`Leu
`47
`Tle
`Met
`Tle
`Met
`48
`
`
`
`
`60 Ala Ser Ala Ala
`
`Gly
`Thr
`Ser
`Arg
`Met
`Arg
`Val
`Ne
`Phe
`Tyr
`Phe
`Gly
`
`Tyr
`Lys
`Thr
`Lys
`He
`Lys
`Ala
`Leu
`Val
`His
`Tyr
`Arg
`
`Tyr
`Lys
`Thr
`Arg
`Met
`Arg
`Ala
`fle
`Phe
`Tyr
`Phe
`Arg
`
`Tyr
`Lys
`Thr
`Lys
`ile
`Lys
`Ala
`Leu
`Val
`His
`Tyr
`Arg
`
`* Numbers correspond to those in Figure 1.
`
`

`

`HUMANIZED ANTI-T CELL ANTIBODIES
`
`4369
`
`thesized. The final DNA sequences of BMA 031-EUCIV2
`VH and VL,excluding the EcoRI and Hindiil cloning ends,
`are shownin 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 EcoRI-HindiIll
`fragments consisting of 10 to 15 overlapping (10-15
`nucleotide overlap) oligomers (75-110 nucleotides). The
`oligomers were phosphorylated, annealed andligated into
`a pUC vector previously cut with EcoRI and HindiIll. The
`assembled fragments were sequenced to verify accuracy
`of synthesis.
`Reconstruction of BMA 031 genomicfragments with
`humanized V exons. To increase the probability of effi-
`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 Hindlll VL genomic
`fragments of BMA 031 (Fig. 3). Due to the lack of unique
`restriction enzymesites, several subclonings were nec-
`essary. To achievethis goal, four vectors, each containing
`modified genomic subfragments, were constructed. The
`first vector, pUCBMAVH-1.0HAN was constructed by
`subcloning the 1.0-kb HindIII BMA 031 VH fragmentinto
`pUC19 with subsequentdeletion of the 5’-Nsil site. The
`second vector, pPUCAHBMAVH-5.6RAH, was derived by
`cloning the 5.6-kb EcoRI BMA 031 VH fragmentinto a
`pUC19 vector with a previously deleted Hindlll site. The
`5’-HindiIll site of the insert was then deleted to complete
`the construction. The third vector, PUCBMAVL-1.4RH2,
`was constructed by subcloning the 1.4-kb EcoRI-Hincll
`BMA 031 VL fragment into pUC19. The fourth vector,
`pUCARSBMAVL-3.0H, was made by cloning the 3.0-kb
`HindIII 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-
`quencesinto the genomic fragmentsis outlined in Figure
`4. The newly synthesized Saul-Nsil BMA 031-EUCIV2
`&
`
`VH fragment wasisolated from the pUC19 subclone and
`cloned into PUCBMAVH-1.0HAN. Then,
`the 1.0-kb
`Hindlll
`fragment was
`isolated and cloned into
`PpUCAHBMAVH-5.6RAH. Finally, the 5.6-kb EcoRI frag-
`ment was isolated and subcloned into the mammalian
`expression vector containing the human y 1 C region and
`the gpt geneforselection (Fig. 5).
`The newly synthesized Saul-Hincll BMA 031-EUCIV2
`VL fragment was isolated and cloned into puUCBMAVL-
`1.4RH2. Then, the 1.4-kb EcoRI-Hincll fragment was
`isolated and cloned into pUCARSBMAVL-3.0H. Finally,
`the 3.0 HindIII fragment was isolated and cloned into the
`mammalian expression vector containing the human x C
`region and the neogeneforselection (Fig. 5).
`The BMA 031-EUCIVS constructs were prepared in the
`same manner as BMA 031-EUCIV2. Replacementoligo-
`mers incorporating the coding changes for BMA 031-
`EUCIV3 were synthesized and cloned into the pUCBMA-
`EUCIV2 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
`031 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 hybridomacells by electroporation andselected in
`media containing both mycophenolic acid and Geneticin.
`Transfectomassecreting 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-
`tion (CIV2 and CIV3), with respect to secretion level and
`growth characteristics, was expandedfor further study.
`The BMA 031-EUCIV2 and -EUCIV3 antibodies were
`partially purified by protein A-Sepharose column chro-
`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
`showedthat the antibodies contained human x and y 1 C
`B
`
`180
`
`Sau I
`CCTCAGGTAACAGAGGG!
`
`+
`
` f
`
`jet v
`
`GTATTATAGGTCTCCTCTATAGGTCTACRCGETTAGAGGTAGCTCGEACTCAOGTAGACA
`QM TQS PSTFLS AS Y
`Tehii1 :
`~~--2-0--$--===n= $=ntnnnnnennennn tienennene
`COSGGACAGAGTCAGCATGACCTGCAGTGCCACCTCANGTGTAAGTTACATGCACTGGTA
`GCCCCTGTCTCAGTGGTACTGGACGTCACEGTGGAGTTCACATICAATGTAGGTGACCAT
`GDRVYTMTcCSATSSYSYRHHAVWY
`Fo 1
`\oa--------CDR-1-----------/
`TeAGchGAMGCEDEGGAAGECTCCEAMAAGACTCATTATCACACATECAMACTOSCTC
`SeteeeeyenseeSen ystewsesEs geeeeengeesesenceeeeES
`AGTCGTCTTCGGECCCTTCCEAGGETTTTCTGACTACATACTGTGTAGGTTTGACCGAAG
`ook PGKAPKRLUMYDTSKLAS
`BssB II
`\nn----+CDR-2------.é
`/
`--~------4---------4-+-0290 --==ne ne ---= 420
`TAGAGTCOCTGOSCECTRCATTGSGAGTGGGTCTGGGACCSAGTTCACGCTCACAATCAG
`ACCTCAGGACEEGOGAAGTAACBFTCACECAGACCCTEGCTEAAGTCCEAGTGTTAGTE
`GV PARFIG
`EF TLTIS§
`a I
`
`300
`
`360
`
`CASCOTECACOAGATEATponoenngrnmenemongenerGanaTAGTAACCCGCT
`GFCGGACGTCOGTETACTAAAGCEATGCAAATGACGTCGTCACCECATCATTCGEGGA
`SLO Po DF «Ty yc oa vs 4
`em E
`NnornnennnnnnCDR:3.awees
`CAOGTTORCTCCHOCTACCAAGCTOGAGATTAAACCTAAGTACACTOTICTCATOTTITT
`---------420- ===3-4anenengeenneedtee 540
`GEGCAAGSEAGERCCATGGT#CCAGCTCTAAETTCOATTCATGTGAAAMGAGTAGAAAAA
`TPGGGTRVE
`-/
`---------4----~----4---~-----4--------4-----ne--- 600
`TEATGTGTAAGACACAGGTTTTCATGTTAGGAGTTAAAGTCAGTICAGAAAATCTIGAGA
`AATACACATTCTGTCTCCAAAAGTACAATCOTCAATTTCAGTCAAGTCTITTAGAAGTOT
`-ine II
`MAATGAGGAGEGCTcarrancacrreat
`---------4---~-----+---------
`629
`TETACCTCCICCCGAGTAATAGTCAACTE
`
`181
`
`161
`
`301
`
`3610
`
`481
`
`S$]
`
`60]
`
`-* I
`CcTCACGTEACAATGACATCACTCTEACATIENCTOTECAGGTGTCCACT‘CTSAGGRCE
`Q
`Q
`GGACTCCACTGTTAGTGTAGATGAGACTGTAAGAGAGAAGT!SexcaceneacaaTCckss
`pee I
`aa 1
`IACTAGTTCAGTCTOGASCAGAGOTEAAGAAGECTOGCAGCTEACTEANGETTICCTGCA
`eae etennbeeenee
`-annnn ent enneneecteee
`FIGATCAAGRCAGACCTGTETCCAATTETTCUGACCETOCAGTEACTTCCAANGEAGEH
`a
`GaAEVKK PGS SY KV S CK
`/Baeti
`oe Tir
`AOCTRCCOGATATARATICATaocrancroarccactactrcasceacceaceresce
`ann penneeee nnent
`AQ] ~--------4=pnent ene
`ASGYKPFTSY¥
`v
`‘TCOGAAGGCCTATATTTAAGTGATCGATACAATACGTGACCCACTCOGTEDETOGACESG
`QaerGca
`<-LOOR-1”<f
`pet
`scTesToGHTOCCKTATATTAMTCCTTACAATCATCTTACTAAGTACAATCAA
`nant
`nn=eensto--=== 240
`1G) ennanenab eeeteen
`TCCCCEAGCTCACCTACCCTATATAATTAGGAATGTTACTACAATGATICATGTTACTCT
`ee, ae ee
`
`120
`
`180
`
`61
`
`Figure 2. DNA sequencesof the V re-
`gions of BMA 031-EUCIV2. A, The BMA
`031-EUCIV2 VH region and B, the BMA
`031-EUCIV2 VL region.
`
`Hennonepeennn
`TN TA YU
`FKGRATITA ODES
`‘hasrrTeCotccoccrattanTécoeacrecTCAGerGATTATGECGGATGTACCTEG
`moSeeyi et II
`TGACAGCCTEACATCTGAGCACACTGCOTECTATTECRSTCCAAGAGGGAGCTACTANG
`---------+---------4---------4-----
`eonntenena-n4--------=)
`ACTOCGGACTCTAGACTCCTETGACGCAAGATAMAGACAGETTFCCETOGATERTAC
`s$LRSEOTAPY FP
`D
`Lacokc=
`/
`Kpn t
`ge oebeneonewe peeweeene:
`------~--4-----~-+--4+---------+-----
`ATTACENCGGGTESGTETACTSGGOLCAAGSTACCTs07cheterensSe
`aaereeGeECAAACAAASGACCEEGGTTECATOGGACCACTGACAGAGAMGTCCACTcA
`GPYYW¥GoaGTLY
`SesewoEssesewessf
`Nsi I
`é
`----2=---4-==nnn 449
`ccT.AACTTCTEOCATTEHMATSCATOEE
`GGATTGAAGAGGGTAAGATTTACGT.“"ACAA
`
`301
`
`3610
`
`421
`
`300
`
`360
`
`420
`
`

`

`4370
`
`HUMANIZED ANTI-T CELL ANTIBODIES
`
`Na ss
`
`P
`
`1.0 kb
`
`SN
`
`1.0 kb
`
`Figure 3. Partial restriction enzyme maps of BMA 031 V regions. A.
`The 5.6-kb EcoRI VH fragmentcontaining the VDJs exon. B, the 3.0-kb
`HindIf VL fragment containing the VJ; exons. H. Hindlll; H2, Hinclh N,
`Nsil, P, PstI; R, EcoRI; S, Saul.
`
`regions. Moreover, the antibodies did not react with anti-
`murine antibodies (data not shown).
`Characterization of humanized BMA 031 antibodies.
`The BMA 031-EUCIV2 antibody bound poorly to T cells.
`In contrast, BMA 031-EUCIV3 showsan identical speci-
`ficity as murine BMA 031. They both bind specifically to
`T cells and show no reactivity toward monocytes, E, or
`granulocytes (data not shown).
`Therelative affinities of murine BMA 031, chimeric
`BMA 031 (human IgG1), and the humanized variants
`were compared by competitive immunofluorescence as-
`
`says. The data shownin Figure 6 indicate that both the
`murine BMA 031 antibody and the previously con-
`structed chimeric BMA 031-G1 antibody block the bind-
`ing of BMA 031-FITC in the same dose-dependent man-
`ner. BMA 031-EUCIV3 was about 2.5 timesless efficient
`than murine BMA 031. BMA 031-EUCIV2 was unable to
`totally block BMA 031-FITC binding, even at concentra-
`tions as high as 50 yg/ml.
`BMA031 has been shown to be poor at mediating ADCC
`using humaneffector cells. To evaluate the ADCC capac-
`ity of the humanized antibodies, we compared them to
`rabbit anti-GH-1 antiserum. This antiserum wasthe best
`of eight rabbit anti-human T cell globulins in ADCC
`capacity. As shown in Figure 7, both the chimeric BMA
`031 antibody and the BMA 031-EUCIV3 antibody were
`very efficient at ADCC. Even at very low effector:target
`cell ratios (Fig. 7A) or extremely low antibody concentra-
`tions (Fig. 7, B and C), the engineered antibodies are
`highly potent at mediating killing of the HPB-ALLcells.
`DISCUSSION
`
`Wehave joined the DNA segments containing the CDR
`from the BMA 031 mAbspecific for the a/8 TCR and the
`FR from the human EU antibody to the DNA segments
`encoding human y-1 and «x C regions. When the human-
`ized genes were introduced into non-Ig producing Sp2/0
`cells, functional humanized antibodies specific for T cells
`were assembied and secreted.
`Functional antibody, however, was dependent on sub-
`stitution of various murine FR amino acids into the hu-
`man FR. Theidentification of important FR amino acids
`in the absence of structural data or computer models is
`difficult but, by careful analysis of antibody sequence
`homologies, it is possible to generate a humanized se-
`quence with a high probability of maintaining Ag bind-
`ing. Our method consists of three parts. First, and pos-
`sibly most important, is starting with the humananti-
`body most homologous to the murine antibody under
`
`HONDS,
`
`NH
`
`BH
`
`
`
`R
`
`Nu
`gt
`
`R
`
`H2
`
`BL
`
`
`
`R
`
`Isolate:
`vector
`
`digest with
`Saul+ Ns!
`
`HACN)S,
`
`NA
`
`\solate
`CH
`
`Isotartevector
`
`Re
`
`HR
`
`digest with
`Saul + Hincil
`
`R
`
`ely
`
`Awitscl
`H2
`
`Figure 4, The cloning scheme to re-
`generate the BMA 031 genomic fragments
`with the humanized V regions. A, Substi-
`tuting the humanized VHregion into the
`5.6-kb EcoRI VH fragment. BH, BMA 031
`
`VH exon; CH, humanized BMA 031 VH
`als
`
`
`
`exon; pl,
`pUCBMAVH-1.0HAN;
` p2,
`
`pUCBMACIVH:_p3, pUCBMACIVH-
`1.0HAN; p4, pUCAHBMAVH-5.6RAH; p5,
`pUCAHBMACIVH-5.6RAH; p6, pSV2gpt-
`huyl; p7, pSV2gpt-BMACIVH-huyl. B,
`Substituting the humanized VL region
`into the 3.0 Hindlll VL fragment. BL, BMA
`031 VL exon; CL, humanized BMA 031
`VL exon; p8, pUCBMAVL-1.4RH2: p9,
`pUCBMACIVL;
`pid,
`pUCBMACIVL-
`1.4RH2; p11, pPUCARSBMAVL-3.0H;plz,
`pUCARSBMACIVL-3.0H; pi3, pSV2neo-
`hux; pi4, pSV2neo-BMACIVL-huk. Re-
`striction enzyme sites identified are: H,
`Hindi; H2, Hincll; N, Nsil, R, EcoRl, S,
`Saul.
`
`HR
`
`H2
`
`Sab
`
`Isolate
`vector
`
`digest with
`EcoRi + Hinctl
`
`HR
`
`H2 A
`
`cu
`
`
`
`\solate
`cH
`
`digest with
`Hindill
`
`Gap
`
`wR
`
`c
`
`
`
`digest with
`EcoRI
`
`yRe
`
`HR
`
`tsolate
`CH
`
`R
`
`isolate
`vector
`
`Isolate:
`vector
`
`lsolate
`ch
`
`H
`
`\solate.
`vector
`
`Hindiill
`
`isolate
`cL
`
`digestwith C)
`
`
`

`

`HUMANIZED ANTI-T CELL ANTIBODIES
`
`4371
`
`a
`:
`8
`é
`
`anti-GH 1
`BMA 034
`BMA 031-G1
`BMA-EUCIV3
`—t— ~=—NK.-activity
`
`0
`
`10
`
`30
`20
`E:T Ratlo
`
`40
`
`50
`
`100 ng/ml
`10 ng/mi
`1 ng/ml
`0.1 ng/ml
`Nk-activity
`
`100 ng/ml
`16 ng/ml
`1 ngml
`0.1 ng/ml
`Nk-activity
`
`9
`
`10
`
`30
`20
`E:T Ratio
`
`40
`
`50
`
`c
`
`10
`
`20
`
`30
`
`40
`
`50
`
`100
`
`80
`
`60
`
`40
`
`20
`
`La
`é
`
`> a
`
`c
`8
`£
`a
`
`Qo
`
`6
`
`E:T Ratio
`
`Figure 7. ADCC capacity of BMA 031 antibodies. The cytolytic capac-
`ity of the BMA 031 antibodies was determined in a 20 h [°'Cr] release
`assay as described in Materials and Methods.A, Lysis in the presence
`{ADCC) or absence (NK activity) of antibody (100 ng/ml). B, Lysis at
`various concentrations of BMA 031-EUCIV3. C, Lysis at various concen-
`trations of chimeric BMA 031.
`
`provides insight into those amino acids that could be
`altered to regain activity.
`The T cell binding data with the humanized BMA 031
`antibodies show the importance of FR aminoacids in Ag
`binding. Inclusion of only the BMA 031 CDR (BMA 031-
`EUCIV1) would, mostlikely, not have been sufficient to
`maintain affinity for Ag. Twelve amino acid substitutions
`were made in the H chain V region to regain binding
`affinity (nos. 27, 28, 30, 38, 48, 67, 68, 70, 93, 94, 95,
`and 98). Of these, six may be more important(nos. 38,
`48, 70, 93, 94, and 95) because they represent changes
`from BMA-EUCIV2, which does not bind well, to BMA
`031-EUCIV3, which does bind well. Similarly, for the L
`chain V region, five amino acid substitutions were made
`(nos. 21, 46, 47, 48, and 60). Of these, three (nos. 21, 47,
`and 48) were made from BMA 031-EUCIV2 to BMA 031-
`EUCIV3 and thus may be more important.
`
` 3
`
`
`
`&5
`
`.
`-!
`=
`
`s £
`
`.
`
`R
`
`B
`
`H
`
`R
`
`B
`
`BMA 031-BUCIVH
`
`ei}
`
`fe} as}
`at
`BUMAN GAMMA 1
`
`force
`pSV2-gpt
`
`H
`
`B
`
`e
`
`R
`
`H
`
`BMA 031-EUCIVL
`
`HUMAN KAPPA
`
`pSV2-neo
`
`Figure 5. Expression vectors for humanized BMA 031 V regions. A,
`The H chain expression vector containing the humanized BMA 031 VH
`region, the human y 1 C region, and the guanine phosphoribosyl trans-
`ferase genefor 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.
`
`Media
`BMA 031
`BMA 031-G1
`BMA-EUCIV2
`BMA-EUCIV3
`
`ah O41
`
`600
`
`500
`
`400
`
`300
`
`200
`
`100
`
`>
`
`2=
`
`=
`
`8
`5
`$
`a/-
`9°
`=
`
`A
`
`1
`
`10
`
`100
`
`Antibody (ug/ml)
`Figure 6. Relative affinities of BMA 031 antibodies. Competitive im-
`munofluorescence assays with the BMA 031 antibodies, HPB mononu-
`clear cells and BMA 031-FITC (2 ug/ml) were performed as outlined in
`Materials and Methods.Intensity of fluorescence is expressed as mean
`channel number.
`
`study. This effectively limits the number of amino acid
`differences that must be addressed. Second, because the
`assignment of CDR is based on homology and not func-
`tion, the choice of maintaining the murine sequence on
`either side of the CDR is important. Evidence is emergin