0022—l767/91/14712-4366802.00/0
`THE JOURNAL or 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 01/6 T CELL RECEPTOR
`
`CLYDE W. SHEARMAN,” DAN POLLOCK,* GARY WHITE,* KATHY HEHIR,‘
`GORDON P. MOORE,“ E. J. KANZY,‘AND ROLAND KURRLE”
`
`From the *Genzyme Corporation, Framingham, MA 01701; and 'Behringwerke Aktiengesellschaft. D-355O
`Marburg, West Germany
`
`Completely humanized antibodies with specificity
`for the human a/B 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 was significantly 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.
`
`mAb are emerging as a major modality for therapy of
`various pathologic conditions including malignant dis-
`ease. cardiovascular disease, and autoimmune diseases.
`Some of these have demonstrated efficacy in treating
`colon carcinoma (1). B cell lymphomas (2), neuroblastoma
`(3), and in preventing transplant rejection [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
`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 publication of this article were defrayed in part by the
`payment of page charges. This article must therefore be hereby marked
`advertisement in accordance with 18 U.S.C. Section 1734 solely to indi-
`cate this fact.
`1 Address correspondence and reprint requests to Dr. C. W. Shearman.
`Sterling Drug. Inc. 512 Elmwood Avenue. Sharon Hill. PA 19079.
`2 Present address Department of Molecular Genetics, SmithKline Bee-
`cham. 709 Swedeland Road, King of Prussia. PA 19406.
`
`Chimeric antibodies may be effective in lowering the
`HAMA response in patients and increasing serum half-
`lives, but these properties are still inferior to human
`3 Abbreviations used in this paper: HAMA, human anti-murine anti-
`body; ADCC, antibody dependent cell mediated cytolysis; FR. framework
`region.
`4366
`
`Pfizer v. Genentech
`
`tive mAb therapy may require frequent multiple treat-
`ments with large amounts of murine antibody. Second,
`administration of murine IgG elicits a brisk HAMAa re—
`sponse that that can further reduce the circulating half-
`life of the mAb and produce allergic reactions including
`anaphylaxis (8—10].
`Almost all of the murine 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 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
`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-
`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 hybridomas are difficult to prepare. are often unsta-
`ble, and secrete antibody at low levels (14, 15). The mAb
`generated are usually of the IgM class and of 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 human regions (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 HAMA response that alters its pharmacoki-
`netics. antibody responses to chimeric 171A have been
`dramatically reduced. Moreover. the circulating half-life
`was increased 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.
`
` Pfizer v. Genentech
`IPR2017-01489
`Genentech Exhibit 2033
`
`IPR2017—01489
`
`Genentech Exhibit 2033
`
`

`

`HUMANIZED ANTI-T CELL ANTIBODIES
`
`4367
`
`antibodies. Inasmuch as chimeric antibodies are still 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 human C regions should be essentially human. This
`technology, although technically straightforward, is not
`always totally 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).
`We report here the production of a humanized anti-
`body, without the use of sophisticated structural data,
`which retains the affinity and Specificity of BMA 031, a
`murine mAb directed against the human a/B 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/O-Agl4 hybridomas were
`cultured in DMEM media supplemented with 10% FCS. 2 mM L-
`glutamine. 10 mM HEPES, pH 7.3. 10 mM nonessential amino acids
`(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 Itng xanthine. and 500
`pg/ml Geneticin (GIBCO). All lines were maintained at 37°C in 7%
`C02.
`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
`DNA synthesizer. Each region was synthesized completely as EcoRI-
`HindIII fragments consisting of overlapping (10—15 nucleotide over-
`lap) oligomers (75—1 10 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 thdlll.
`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 V regions were inserted into previously isolated
`genomic fragments (24) in place of the murine V regions. The re-
`sulting 5.6-kb EcoRl VH fragment was cloned into a mammalian
`expression vector containing the human 71 C region and the gpt
`gene for selection. The 3.0-kb thdlll VL fragment was cloned into
`a vector containing the human K C region and the neo gene (see Fig.
`5).
`
`Transfection of DNA into Sp2/0 cells by electroporation. DNA
`was introduced into murine hybridoma Sp2/O-Ag14 cells by electro-
`poration. The l to 2 X 107 actively growing Sp2/0-Agl4 cells were
`washed and resuspended in 1.0 ml of sterile PBS. A total of 15 pg of
`each humanized, ng 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/cm electric pulse was delivered for 10 ms (Transfector 300.
`BTX. San Diego, CA). After shocking, the DNA/cell mixture was
`returned to ice for 10 min and then diluted in 40 ml of supplemented
`DMEM and incubated at room temperature for 10 min. Finally. the
`cells were transferred to a 37°C incubator with 7% C02 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 104 cells/well.
`Cytofluorometric assayfor affinity. To analyze the relative 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 l h in the dark with mAb at various
`concentrations (0.05—50 ug/ml) premixed with either FlTC-BMA031
`or FlTC-BMAEUCIVB (2 ug/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 FACStar Plus software and
`is expressed as mean channel number.
`Cytotoxicity assays. To measure the cytolytic capacity of the
`BMA 031 antibody preparations, a 20 h [5‘Cr] release assay was
`performed to measure ADCC and NK activity. [5‘Cr]-labeled HPB-
`ALL target 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/B 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 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 described earlier (33). Spontaneous
`[SICr] release in the absence of effector cells and in the presence of
`the antibodies being tested was always less than 5%. All samples
`were analyzed in triplicate.
`
`RESULTS
`
`Designing humanized BMA 031 antibodies. To deter-
`mine the 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 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-
`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-EUCIV1 (Fig. l).
`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 a1. (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 O31-EUCIV2
`(Fig. 1). Additionally, all differences within four amino
`acids of the CDR could be maintained murine. We refer
`
`to this antibody as BMA 031-EUC1V3.
`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 enhance their 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 0VOLVOSGAEVKKPGSSV'KVSCKASGGTFSRSAIIWRQAPGQGLEVHGGIVPHFGPPNY
`BMA
`E
`0
`P LV
`A
`H
`YK TSYVHH K K
`I Y N YNDVTK
`CIV-l
`SWHII
`Y N YNDVTK
`CIV—Z
`YK TSYVHE
`Y N YNDVTK
`CIV-S
`YK ’I'SYVHH
`I Y N YNDVTK
`\CDR/
`\ -----CDR-2
`1
`
`110
`90
`70
`EU AOKFOGRVTITADESTNTAYHELSSLRSEDTAFYPCAGG . YGIYSPEEY . . NGGLVTVSS
`BHANEKKALSKSS
`T
`SVHYRSYDDGFVVGQT
`A
`CIV—l NE K
`5 YD DGFV VGQ T
`CIV—Z
`NE K A
`R 5 YD DGFV VGO T
`CIV-3
`NE
`K KA L
`VHY R 5 YD DGFV V60 T
`-----/
`\——CDR-3——/
`
`K
`
`B
`
`
`
`50
`30
`10
`EU DIOHTOSPS'I’LSASVGDRV'I'ITCRASOSINTVLAVYQOKPGKAPKLLHYKASSLESGVPS
`BMA OVL
`AIH
`PEK H
`STSV-SYHH
`STS RVIDTKA
`A
`CIV—l
`S TS V.SYHH
`Dr K A
`CIV-Z
`S TS V.SYIIE
`01' K A
`R
`CIV-3
`5 TS V.SYHH
`RVI DT K A
`\——CDR-1——/
`\CDR—Z/
`
`A
`A
`
`H
`
`90
`70
`EU RFIGSGSGTEPTLTISSLQPDDFATYYCQQYNSDSKMFGQGTKVEVK
`BMA
`S
`SYS
`HEAR A
`US NPLT
`A
`L L
`CIV—l
`VS NPLT
`G
`I
`CIV-Z
`VS NPLT
`G
`I
`CIV—3
`VS NPLT
`G
`I
`\—CDR-3—/
`
`Figure 1. 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
`
`a
`
`EU AA
`AA Position
`H chain. EU specific
`70
`Ile
`72
`Ala
`74
`Glu
`93
`Phe
`95
`Phe
`98
`Gly
`
`L chain. EU specific
`1 0
`Thr
`48
`Met
`63
`Ile
`70
`Glu
`81
`Asp
`
`BMAO31
`AA
`
`Mouse
`AA
`
`"
`5
`"
`Val
`Tyr
`Arg
`
`Ser
`Ile
`Ser
`Asp
`Glu
`
`Leu
`Ser
`Lys
`Val
`Tyr
`Arg
`
`Ile
`Ile
`Ser
`Ser
`Glu
`
`Leu
`Val
`Lys
`Val
`Tyr
`Arg
`
`lie
`lie
`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
`Gln
`Pro
`Ser
`Ser
`Ala
`Pro
`Ala
`Leu
`Met
`Val
`Arg
`Lys
`Ala
`Val
`Ser
`°
`Gln
`Glu
`Glu
`Tyr
`His
`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 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 1 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 amino acids of the CDR (nos.
`
`70. 95, and 98], and these are addressed in BMA-031-
`EUCIVS. In one position (no. 93) the human consensus
`sequence is the same as BMA 031. Moreover, the Phegg
`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 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. 10 and 70) were also not changed to limit
`the number of substitutions. There are eight BMA O31
`specific amino acids in the H chain. In two positions (nos.
`7 and 82) the BMA 031 sequence is the same as EU. l-Iis94
`is unique to BMA 031. This position is considered “invar~
`iant" with Tyr94 occurring more than 98% of the time.
`Therefore, we decided to incorporate this change into
`BMA 031—EUCIV3. The remaining five positions (nos. 1,
`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 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 of 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-
`where else. 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 enzyme sites 5’ and 3’ of the coding region of BMA
`031 were identified and this flanking sequence was in-
`corporated into the final humanized sequence to be syn—
`TABLE 1]
`
`Amino acid (AA) changes in EU FR
`a
`BMAOSI
`CIV2
`CIVS
`
`AA Position
`EU AA
`AA
`AA
`AA
`H chain
`27
`28
`30
`38
`48
`67
`68
`70
`93
`94
`95
`98
`L chain
`Met
`Ile
`Met
`Ile
`21
`Arg
`Arg
`Arg
`Leu
`46
`Trp
`Leu
`Trp
`Leu
`47
`lie
`Met
`Ile
`Met
`48
`
`
`
`
`AlaScr60 Ala Ala
`
`Gly
`Thr
`Ser
`Arg
`Met
`Arg
`Val
`Ile
`Phe
`Tyr
`Phe
`Gly
`
`Tyr
`Lys
`Thr
`Lys
`Ile
`Lys
`Ala
`Leu
`Val
`His
`Tyr
`Arg
`
`Tyr
`Lys
`Thr
`Arg
`Met
`Arg
`Ala
`Ile
`Phe
`Tyr
`Phe
`Arg
`
`Tyr
`Lys
`Thr
`Lys
`Ile
`Lys
`Ala
`Leu
`Val
`His
`Tyr
`Arg
`
`‘1 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 Hindlll cloning ends,
`are shown in Fig. 2.
`Synthesis of humanized BMA 031 V regions. The L
`and 1-1 chain V region exons encoding the humanized
`antibodies were synthesized completely as EcoRl-Hindlll
`fragments consisting of 10 to 15 overlapping (10—15
`nucleotide overlap) oligomers (75—1 10 nucleotides). The
`oligomers were phosphorylated, annealed and ligated into
`a pUC vector previously cut with EcoRI and HindIIl. The
`assembled fragments were sequenced to verify accuracy
`of synthesis.
`Reconstruction of BMA 031 genomic fragments 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 EcoRl VH and 3.0-kb HindIII VL genomic
`fragments of BMA 031 (Fig. 3). Due to the lack of unique
`restriction enzyme sites. several subclonings were nec-
`essary. To achieve this 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 fragment into
`pUC19 with subsequent deletion of the 5’-NsiI site. The
`second vector, pUCAHBMAVH-5.6RAH, was derived by
`cloning the 5.6-kb EcoRI BMA 031 VH fragment into a
`pUC19 vector with a previously deleted Hindlll site. The
`5’-HindIII 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 EcoRl-HincII
`BMA 031 VL fragment into pUC19. The fourth vector,
`pUCARSBMAVL-BDH, 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 Sail
`site in the polylinker.
`The cloning scheme to replace the humanized se-
`quences into the genomic fragments is outlined in Figure
`4. The newly synthesized Saul-Nsil BMA 03l-EUCIV2
`A
`
`VH fragment was isolated from the pUClQ subclone and
`cloned into pUCBMAVH-1.0HAN. Then,
`the 1.0-kb
`HindIII
`fragment was
`isolated and cloned into
`pUCAHBMAVH-5.6RAH. Finally. the 5.6-kb EcoRI frag-
`ment was isolated and subcloned into the mammalian
`
`expression vector containing the human 7 l C region and
`the gpt gene for selection (Fig. 5).
`The newly synthesized Saul-Hind] BMA 031—EUCIV2
`VL fragment was isolated and cloned into pUCBMAVL-
`l.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 K C
`region and the neo gene for selection (Fig. 5).
`The BMA 031-EUCIV3 constructs were prepared in the
`same manner as BMA 031-EUCIV2. Replacement oligo-
`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 hybridoma cells by electroporation and selected in
`media containing both mycophenolic acid and Geneticin.
`Transfectomas secreting humanized BMA 03 1 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 expanded for 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
`showed that the antibodies contained human x and -y 1 C
`3
`
`Figure 2. DNA sequences of the V re—
`gions of BMA 031-EUCIV2. A, The BMA
`031-EUC1V2 VH region and B. the BMA
`031—EUCIV2 VL region.
`
`San I
`/
`C(,TGAGGTGACAATGACATCI'ACI'CI‘GACAHCI‘CTCKTCAGGTGTCCACI'LTCAGGTCC
`
`GGACI'CCACTGTK'ACTGTAGATGAGACTGTAAGAGACAAEK'CCACAGGTGAGAGTCCAGG
`(l
`V
`0
`Spa I
`See I
`i.
`/
`/
`AACTAGU'CAGT£1MASCAGAGGTTAAGAAGCCTGGGAGHCAGTGAAGCH'I‘CCTGCA
`........................................._.............-._
`TTGATCAAGTCAGACCCCGTCl'CCAATTmCGGACCL-I‘CGAGTCACITCCMAGGACGT
`LVOSGAEVKKPGSSVK‘ISCK
`kph 11
`Du III
`/
`/
`AGGCI‘TCCGGATATAAATTCACTAGUATG'ITATGCACTGGGTGAGGCAGGCACCI'GGCC
`_______________________________________________________
`TCOGAAGGCCTATAT'H'AAGTGATCGATACAATACGTGACCCACI‘COGTCCGTMACCGG
`ASGYKPTSYVKHVVROAPGQ
`Xho I
`\.-—cnn-i_-./
`/
`AGGGGCTCGAGTGGATGGGATATAT'I'AATCCT'I'ACAATGATCH’ACI‘AAGTACAATGAGA
`_________«0..................-...............-........-..
`TCCCCGAGCI'CACL'IACCUATATAATI'AGGAATGTTACTACAATGATTCATG‘H‘ACI'LT
`GLEVHGYINPYNDVTKYNBK
`\.................cnR.z—————————————————
`XII XI!
`/
`AG'ITCAMGGCAGGGCCACM11AOGGCCGACGAGTCCACTMTACAGCCI'ACATGGAGC
`.........................................................
`rcucmcccrccoccrarrAA'chcccchcTCAGcrcArrArc'tcoGAi‘MAccrcc
`FKGRATITADESTNTAYHBL
`......../
`351 II
`/
`TGAGCAGCCI‘GAGATCTGAGGACACYGCGTK'CI‘AchGTGCMGAGGGAGCMC‘MTG
`........................................................
`Acres-resume!Ax;Acrcac'rcAcccAAcArAAAcACAccri-crcccrocnaAna
`SSLRSEDTAPYPCAIGSYYD
`\......CDR-S-
`Kpn i
`/
`ATMCGACGGG‘H'!G‘mACEGGGGCCAAGGI‘ACCCTGG‘l‘CACrGICTmCAGGTGAGT
`_______________________________________________________
`TAATGCfGCCCAAACAAA‘NACCGCGGH'CCATGGGACCAI-TGACAGAGAACICCACTCA
`YDGIVYVGOGTLVTVSS
`—————————————————I
`N51 I
`/
`CCTAACKTCTCCCA‘HCIAAATGCATG'H‘
`GGATTGAAGAGQETAAGATTI‘AGGTACAA
`
`60
`
`120
`
`180
`
`240
`
`300
`
`360
`
`£10
`
`61
`
`121
`
`18!
`
`231
`
`301
`
`361
`
`611
`
`Sun I
`I
`CCI'CAGGTAACAGAGGG
`
`
`
` Sunk V
`_______________________________________________________
`GTAn‘ATAcc'i‘crccrcrumrrcucrmmmmmemnuocumu
`DIQHTOSPSTLSASV
`Tthlil r
`/
`CGGGGACAGAGK'CACCATGACCIGCAGTGCCACCICMGTGTMHACATGCACEGGTA
`_______________________________________________________
`Gcccc'rcrcrcmmcncramcmCAOGGTGGAGHCACAITCAAMACGTGAOCAT
`GDRVTHTCSATssvsrnav‘i
`An I
`\ ..........amt—1.........../
`/
`TCAGCAGAAGCCCGGGAAGGCICCCMAAGACI‘GATGTATGACACATCCMACI‘GGCH'C
`________________________________________________________
`Acrccrcn‘cccscccrrcccAcccrrncrcAcrACArAnmercchcccAAa
`QOKPGKAPKRLNYDTSKLAS
`3553 n
`\.......0111.2______x
`/
`rccAcrcccrcmocmnccammrmGMchAmuccnacumAc
`_______________________________________________________
`AcmCAchAccccccAAGrAAcocruccchcccmacrCAAGchcAmnAcrc
`GVPARPICSGSGTEFTLTIS
`5nd I
`/
`CAcccrcCAGcCAGATGAmocci-AocrAnAcmcCAcCAchGAG'rAa-rAAccoccr
`_______________________________________________________
`GTCGGACGA'CGGK‘CIACTAAABCGATGCATMTGACGGTOG’I’CACCI‘CATDA‘H‘GGGCGA
`SLOPDDYATYYCOOVSSNPL
`\————————————coma»---
`Kpn I
`/
`CACGTTEGGTOGAWACCMGGTCGAGATTAMCGTMGTACACI'HTCICATL‘HTH
`.........................................................
`GEGCMGCCACCICCATGGT!CCAGCTG'AA‘HTGCATTCATGTGAAMGAG‘AGAAAAA
`T
`17
`G
`G
`G
`T
`K
`V
`E
`I
`K
`—/
`'1'1'ATGTGTAAGACACAGG'KTTICATGTK‘AGGAGTK‘AAAGTCAGTTCAGAAAATCTIGAGA
`...........-....._........__H.._______.-_....._..._____---
`AA'I'ACACAHCK'GTGFCCAAAAGTACAATCCImmCAGK'CAAGTCmTAGAAflfl
`Einc II
`/
`AAATGGAGGAGGGCICA’ITATCAGH‘GAC
`
`180
`
`2‘0
`
`300
`
`360
`
`£20
`
`680
`
`5‘0
`
`600
`
`mACUCéCCCGAGTAAIALfi‘CAAflG
`
`181
`
`141.
`
`301
`
`361
`
`421
`
`491
`
`5101
`
`60]
`
`

`

`4370
`
`HUMANIZED ANTI-T CELL ANTIBODIES
`
`R
`
`B
`
`H
`
`H
`
`EN
`
`5
`
`NB SS
`
`P
`
`R
`
`1.0kb
`
`R
`
`S
`
`P
`
`1121’
`
`SN ll
`
`VJ5
`
`1.0 kb
`
`Figure 3. Partial restriction enzyme maps of BMA 031 V regions. A.
`The 5.6-kb EcoRI VH fragment containing the VDJS exon. B, the 3.0-kb
`thdlll VL fragment containing the Vds exons. H. Hindlll; H2, Hincll; N.
`NsiI; P. Pstl; R, EcoRl; 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 shows an 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).
`The relative affinities of murine BMA 031, chimeric
`
`BMA 031 (human IgGl), and the humanized variants
`were compared by competitive immunofluorescence as-
`
`says. The data shown in 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 times less efficient
`than murine BMA O31. BMA 03l-EUCIV2 was unable to
`
`totally block BMA 031-FITC binding, even at concentra—
`tions as high as 50 pg/ml.
`BMA 031 has been shown to be poor at mediating ADCC
`using human effector cells. To evaluate the ADCC capac-
`ity of the humanized antibodies, we compared them to
`rabbit anti-GH-l antiserum. This antiserum was the 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 effectorztarget
`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-ALL cells.
`DISCUSSION
`
`We have joined the DNA segments containing the CDR
`from the BMA 031 mAb specific for the a/{i TCR and the
`FR from the human EU antibody to the DNA segments
`encoding human 7-1 and K C regions. When the human—
`ized genes were introduced into non-1g producing Sp2/O
`cells, functional humanized antibodies specific for T cells
`were assembled and secreted.
`
`Functional antibody, however, was dependent on sub-
`stitution of various murine FR amino acids into the hu-
`
`man FR. The identification 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 human anti-
`body most homologous to the murine antibody under
`
`H(N)S
`
`N H
`
`
`
`R
`
`N H
`
`i?
`
`H2
`
`
`
`R
`
`H2H
`
`Figure 4. The cloning scheme to re-
`generate the BMA 031 genomic fragments
`with the humanized V regions. A. Substi-
`tuting the humanized VH region into the
`5.6-kb EcoRI VH fragment. BH. BMA 031
`VH exon; CH. humanized BMA 031 VH
`exon;
`p1,
`pUCBMAVH-IOHAN;
`p2.
`pUCBMACIVH:
`p3.
`pUCBMACIVi—i-
`1.0HAN; p4. pUCAHBMAVH-5.6RAH: p5.
`pUCAHBMACIVH-5.6RAH; p6. psvzgpt-
`huvl: p7. pSVngt-BMACIVH-huyl. B.
`Substituting the humanized VL region
`into the 3.0 Hindlil VL fragment. BL. BMA
`031 VL exon: CL. humanized BMA 031
`VL exon: p8, pUCBMAVL-IARHZ p9.
`pUCBMACIVL:
`p10.
`pUCBMACiVL—
`1.4RH2:p11. pUCARSBMAVL-3.0H; p12.
`pUCARSBMACiVL—3.0H: pi3. pSV2neo—
`hux; p14, pSV2ne0vBMACIVL-hux. Re-
`striction enzyme sites identified are: H.
`Hindlll; H2. HincII: N, Nsil: R. EcoRl; S.
`Saul.
`
`o digs."with a
`A“
`CH
`NH
`
`Soul 6 Nil
`
`H(N>$
`
`lsoims
`vaciov
`
`V
`
`“Him”
`Isolate \
`C”
`m )
`
`H n
`
`e
`
`R
`
`
`
`.0 digestmm a
`
`EcoRI
`
`Isolate
`vecto!
`
`isolate
`H
`C
`
`o dices?MM 0
`
`Saul + Hlncll
`
`isolate
`CL
`
`Isolde
`vector
`
`H R
`
`
`R(
`
`Q1..
`
`
`a
`
`Isolate
`vector
`
`R
`
`H2
`
`H R
`
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`
`a
`
`ISOIOVD
`CL
`
`n
`
`Isolate
`vector
`
`digestwith
`
`5Com Q W36“
`
`H n
`
`HZH
`
`CL
`
`
`
`digestmm a
`
`Hlndlll
`
`lsolma
`CL
`
`H
`
`.a
`
`Isolate
`vector
`
`m)
`
`HR
`
`
`
`

`

`HUMANIZED ANTI-T CELL ANTIBODIES
`
`4371
`
`100
`
`
`
`
`80
`
`'3
`3 so
`
`4o
`
`20
`
`O
`
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`‘
`3
`&
`
`fl
`1.4
`‘c‘
`
`o g
`
`n.
`
`R
`
`n
`
`H
`
`R
`
`B
`
`II” ml
`
`mm 031—sumo;
`
`sum cum 1
`
`pSVZ—gpt
`
`H
`
`B
`
`a
`
`R
`
`H
`
`mm 031—EUCIVL
`
`HUMAN um
`
`pSVZ—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 7 1 C region. and the guanine phosphoribosyl trans—
`ferase gene for selection. B, The L chain expression vector containing the
`humanized BMA 031 VL region, the human x C region and the neomycin
`resistance gene for selection.
`
`600
`
`O
`
`1 0
`
`3 0
`2 0
`ET RItlo
`
`4 0
`
`5 0
`
`0
`
`1 0
`
`3 0
`2 0
`ET Ratlo
`
`4 O
`
`5 0
`
`C
`
`anti-GH 1
`BMA 031
`
`BMA 031-61
`.
`.
`BMA-Eucwa
`NK-actiwiy
`
`100 ng/ml
`10 ng/ml
`
`1 ng/ml
`0.1 ng/ml
`NK-activity
`
`100 ng/ml
`10 ng/ml
`1 nglml
`
`0.1 ng/ml
`NK-activiiy
`
`100
`
`80
`
`D 3
`
`>.
`" 6°
`E
`°
`E
`o.
`
`40
`
`20
`
`Media
`BMA031
`BMA031-G1
`BMA-EUCIV2
`BMA-EUCIV3
`
`u.
`
`500
`
`400
`
`30°
`
`200
`
`100
`
`.>.-
`
`3 #
`
`2
`-
`
`3
`5
`3
`0h
`
`o 3
`
`.01
`
`.1
`
`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 O31-FITC (2 (mg/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 emerging
`that the “functional” CDR loops can be displaced from
`the “Kabat" CDR by as many as five amino acids. Kabat
`et al. (34) places CDR-1 of the H chain V region from
`amino acids nos. 31 to 35 whereas crystal structure
`shows the loop to be from residues nos. 26 to 32 (35).
`Third, the identification of potentially “Ag specific” amino
`acids in both the human and murine antibody may be
`important. Although the identification may be straight-
`forward, prioritizing their importance is very difficult.
`lnasmuch as the goal is to produce the most human—like
`sequence. these changes must be kept at a minimum.
`Our decision to keep similar amino acids human and only
`change the more unusual amino acids turned out to be
`correct in t